Complex Comprising A Cell Penetrating Peptide, A Cargo And A TLR Peptide Agonist For Treatment Of Colorectal Cancer

ABSTRACT

The present invention provides a novel complex for use in the prevention and/or treatment of colorectal cancer, the complex comprising a) a cell penetrating peptide, b) at least one antigen or antigenic epitope, and c) at least one TLR peptide agonist, wherein the components a)-c) are covalently linked. In particular, compositions for use in the prevention and/or treatment of colorectal cancer, such as a pharmaceutical compositions and vaccines are provided.

The present invention relates to the field of vaccination, in particularto vaccines for the prevention and/or treatment of colorectal cancer(CRC).

Colorectal cancer (CRC) Globally, CRC is a common and lethal disease andthe third most commonly diagnosed cancer, third in males and second infemales, with more than 1.36 million new cases and about 694,000 deathsoccurring in 2012. The risk of developing CRC is influenced by human,environmental and genetic factors (Cancer, I.A.f.R.o. GLOBACAN 2012.2012 [cited 2015 Jul. 5]; Available from:globocan.iarc.fr/Pages/burden_sel.aspx). Although the CRC incidence ismore predominant in man than in woman, it is more significantly impactedby the age and life style. Indeed over 90% of patients diagnosed withCRC are 50 years old or more (Prevention, C.f.D.C.a. What Are the RiskFactors for Colorectal Cancer? 2015 [cited 2015 Jul. 5]) while 2/3 ofthe cases occurred in developed countries. There is evidence thatconsumption of meat in general and more specifically red meat orconsumption of alcoholic beverages that are leading to general obesity,are significantly increasing the risks of CRC (Stewart, B. and C. Wild,World Cancer Report 2014, B. Stewart and C. Wild, Editors. 2014,International Agency for Research on Cancer: Geneva). Two other factorsare significantly impacting the risks of developing colorectal cancer:hereditary and inflammatory bowel disease. Familial adenomatouspolyposis (FAP) is for example significantly increasing the risks of CRCfor less than 50 years old people (Burt, R. W., J. A. DiSario, and L.Cannon-Albright, Genetics of colon cancer: impact of inheritance oncolon cancer risk. Annu Rev Med, 1995. 46: p. 371-9). FAP, like MAP(MUTYH-associated polyposis) (Sieber, O. M., et al., Multiple colorectaladenomas, classic adenomatous polyposis, and germ-line mutations in MYH.N Engl J Med, 2003. 348(9): p. 791-9) or Lynch syndrome (Lynch, H. T.,et al., Genetics, natural history, tumor spectrum, and pathology ofhereditary nonpolyposis colorectal cancer: an updated review.Gastroenterology, 1993. 104(5): p. 1535-49), is associated with genemutations that predispose patients to inheritance of multiple colonicadenomas (Dennis J Ahnen, D. M., F A. Colorectal cancer: Epidemiology,risk factors, and protective factors. 2015 [cited 2015 Jul. 9];Available from:www.uptodate.com/contents/colorectal-cancer-epidemiology-risk-factors-and-protective-factors).The influence of diseases such as ulcerative colitis or Crohn's diseasein developing a CRC is well documented. Ulcerative colitis impact is forexample linked to a 3 to 15-fold increase in risk (Ekbom, A., et al.,Ulcerative colitis and colorectal cancer. A population-based study. NEngl J Med, 1990. 323(18): p. 1228-33). Although there are less data forCrohn's disease, similar relative risks are associated with thisdisease. For patients having family history in terms of CRC, adenomatouspolyposis or inflammatory bowel disease, early screening is stronglyrecommended (Jemal, A., et al., Global cancer statistics. CA Cancer JClin, 2011. 61(2): p. 69-90).

Like in many cancers, the CRC patient's survival time depends on diseasedevelopment stage. Patients with early stages (Stage 0, I and II) havegood prognosis (more than 75%). Stages III have a more heterogeneoussurvival rate (from 90% down to 50%) depending on the tumor invasion onperipheral tissues. Finally, only stage IV is showing a fast andsignificant effect on the patient survival time: Only about 10% ofpatients are surviving more than 60 months after diagnosis.

Existing treatments are generally based on a surgery followed or not bywell-established chemotherapy, radiotherapy and/or targeted therapy(Moertel, C. G., Chemotherapy for colorectal cancer. N Engl J Med, 1994.330(16): p. 1136-42; Meyerhardt, J. A. and R. J. Mayer, Systemic therapyfor colorectal cancer. N Engl J Med, 2005. 352(5): p. 476-87). Dependingon the disease progression stage, existing treatments allow a 30% to 95%survival rate two years after diagnosis. All early stage CRC treatmentstrategies are initiated with surgery, combined or not with additionalregimen. The decision on the follow-up therapy regimen depends on thedisease progression stage identified during the preliminary screeningresults. For advanced CRC, the use of chemotherapy or targeted therapyas first line treatment is generally recommended. The most commonly usedregimens are FOLFOX, CapeOX or FOLFIRI. These treatments may be used incombination with one of the following recommended biological drugstargeting VEGF (Genentech bevacizumab/Avastin® or Regeneron-Sanofiaflibercept/Zaltrap®) or EGFR (Merck-Serono cetuximab/Erbitux®). Othertargeted therapies could be proposed as stand-alone first linetreatment. Amgen's EGFR monoclonal antibody (panitumumab/Vectibix®) and,more recently, Bayer's small molecule kinase inhibitor Regorafenib(Stivarga®) have demonstrated their capacity to increase overall CRCpatients' survival time. If the disease already importantly spread toother organs, the VEGF monoclonal antibody Ramucirumab (Cyramza®) fromEli Lilly may be used. For stage IV, radiation may be used to relievesymptoms such as pain.

Although CRC chemotherapy is well established (Moertel, C. G.,Chemotherapy for colorectal cancer. N Engl J Med, 1994. 330(16): p.1136-42) the momentum towards more effective and less subject tosecondary effects strategies importantly evolved in the past decades,namely for stage IV CRC (Gallagher, D. J. and N. Kemeny, Metastaticcolorectal cancer: from improved survival to potential cure. Oncology,2010. 78(3-4): p. 237-48). The first trials were directed towardscomplementary adjuvant therapy. However after many years, the value ofpostoperative 5-FU based therapy remains controversial, in particularfor patients with stage II CRC (Meyerhardt, J. A. and R. J. Mayer,Systemic therapy for colorectal cancer. N Engl J Med, 2005. 352(5): p.476-87).

In that context, immunotherapies have been carefully evaluated. Theimmune system can recognize and to some extent eliminate tumor cells,however, this anti-tumor response is often of low amplitude andinefficient. Boosting this weak anti-tumor response with therapeuticvaccination has been a long sought goal for cancer therapy. Modulatingthe immune system to enhance immune responses has thus become apromising therapeutic approach in oncology as it can be combined withstandard of care treatments.

Promising preclinical data and advances in clinical trials, includingthe recent FDA approval of the Sipuleucel-T vaccine and of theanti-CTLA-4 antibody, show that active immunization is a safe andfeasible treatment modality for certain cancer types. Induction oftumor-specific cytotoxic T lymphocytes (CTLs) mediated immune responseshas been reported using different approaches including modified tumorcell vaccines, peptide vaccines, recombinant viral vectors, DNA,protein, or dendritic cell vaccines. However, the anti-tumoral immunitymediated by CTLs only occasionally correlates with tumor regression andonly a few projects have reached the phase III clinical stage.

Overall, cancer vaccines showed very limited clinical efficacy so far.Indeed, at the end of 2011, amongst the 300 hundred ongoing cancervaccine clinical trials, only 19 phase III trials were reported(globaldata, 2012). Amongst them, there are NeuVax, a peptide vaccinefor breast cancer, Stimuvax, a liposome based vaccine for Non-Small CellLung Carcinoma (NSCLC) and breast cancer, TG4010, a vaccinia-basedvaccine for NSCLC and GSK1572932A, an adjuvanted liposome for NSCLC.These four cancer vaccines are based on different technologies and havein common that they are targeting one single antigen.

Therapeutic cancer vaccines can be divided into two principalcategories: personalized (autologous) and standardized vaccines, andfurther classified depending on the technology platform. Currentpersonalized vaccines include tumor lysate vaccines as well as dendriticcells based vaccine (hereinafter cell based). For the latter, antigenloading can occur either with a pulse using tumor lysates, ortransfection with RNA extracted from the tumors. In this case, theantigens are tumor specific or associated, but are not clearly defined.Dendritic cells can also be loaded with defined antigens either withpeptide pulse or using a protein such as the Prostatic Acid Phosphatase(PAP) used to engineer the Provenge® vaccine. However, the manufacturingprocess of these cell-based therapies is time-consuming andlabor-intensive while quality standards are difficult to reach andmaintain. Immunomonitoring creates further complications. Moreover, themajority of the autologous cancer vaccines do not allow the identitiesor quantities of antigens used to be controlled, unlike defined andstandardized vaccines.

In contrast to cell-based therapy (APCs, T cells, CARs, lysates),subunits vaccines (protein or peptides) allow the development of astandardized vaccine with an easier production and significantly betterbatch to batch reproducibility that can be administrated to a broadrange of patients. Furthermore, the antigens are fully defined allowingfor better immune-monitoring and reducing the risk of unwanted effectsof vaccine component.

The different approaches which were evaluated in pre-clinical andclinical development include short peptide vaccines (Slingluff C L, Jr.The present and future of peptide vaccines for cancer: single ormultiple, long or short, alone or in combination? Cancer journal2011;17(5):343-50), long-peptide vaccines (Melief C J, van der Burg S H.Immunotherapy of established (pre)malignant disease by synthetic longpeptide vaccines. Nature reviews Cancer 2008;8(5):351-60) and proteins.In contrast to long peptide and protein vaccines, short peptide vaccineshave a very short half-life and can have negative consequences on theimmune response.

For the protein-based vaccines, the results of targeting MAGE-A3 with arecombinant fusion protein-based vaccine have been enthusiasticallyawaited after promising phase II data in metastatic melanoma (Kruit W H,Suciu S, Dreno B, Mortier L, Robert C, Chiarion-Sileni V, et al.Selection of immunostimulant AS15 for active immunization with MAGE-A3protein: results of a randomized phase II study of the EuropeanOrganisation for Research and Treatment of Cancer Melanoma Group inMetastatic Melanoma. Journal of clinical oncology: official journal ofthe American Society of Clinical Oncology 2013;31(19):2413-20) andnon-small cell lung cancer (NSLC)(Vansteenkiste J, Zielinski M, LinderA, Dahabreh J, Gonzalez E E, Malinowski W, et al. Adjuvant MAGE-A3immunotherapy in resected non-small-cell lung cancer: phase IIrandomized study results. Journal of clinical oncology: official journalof the American Society of Clinical Oncology 2013;31(19):2396-403).However, in 2013 the phase III DERMA trial in melanoma (NCT00796445) didnot meet its first co-primary endpoint, followed in 2014 with a stop ofthe phase III MAGRIT study in NSCL (NCT00480025). Despite these verydisappointing clinical results, protein based vaccines undeniablypresent many advantages.

Also in the context of colorectal cancer scientific progresses in tumorimmunology led to a better understanding of anti-tumor immune responsethrough cellular and humoral pathways (Smith, C. L., et al.,Immunotherapy of colorectal cancer. Br Med Bull, 2002. 64: p. 181-200;Koido, S., et al., Immunotherapy for colorectal cancer. World JGastroenterol, 2013. 19(46): p. 8531-42) and helped better identifyingtumor antigens. This progresses opened new perspective forimmunotherapies in CRC. Passive immunotherapies such as antibodies wereproved as the first targeted therapies for CRC treatment. Thanks totheir capacity in being able to interact with tumor growth pathwaythrough the epidermal growth factor receptor (EGFR) or to inhibit thevascular endothelial growth factor (VEGF), antibodies such as cetuximab,panitumumab and bevacizumab received FDA approval for CRC treatmentrespectively in 2004, 2006 and 2009.

Adoptive cell transfer (ACT) therapy clinical trials for CRC haveunfortunately returned poor results so far. Indeed the limited patientpopulation for both non-engineered and engineered T-cells treatmentsseems being a major hurdle. Similarly, secondary effect observed onpatients treated with T-cells fused with chimeric antigen receptors(CARs), failed to demonstrate ACT as a safe and efficient treatment(Koido, S., et al., Immunotherapy for colorectal cancer. World JGastroenterol, 2013. 19(46): p. 8531-42; Xiang, B., et al., Colorectalcancer immunotherapy. Discov Med, 2013. 15(84): p. 301-8). Thus added tousual ACT drawbacks in terms of feasibility and cost and immune responsememory seems blocking ACT option for the time being.

More recently, the positive clinical trial phase III results on patientsurvival for aflibercept, allowed this anti-VEGF fusion protein to beapproved by the FDA for metastatic colorectal cancer (mCRC) (Clarke, J.M. and H. I. Hurwitz, Ziv-aflibercept: binding to more than VEGF-A—doesmore matter? Nat Rev Clin Oncol, 2013. 10(1): p. 10-1). This targetedtherapy approval paves the way for non-antibody immunotherapies. So far,no active immunotherapies and no immuno-modulators have been approvedfor CRC. Structurally, the vast majority of the molecules tested for CRCare either small molecules, in general kinase inhibitors, or antibodies(respectively 52% and 28%).

In general, a therapeutic cancer vaccine is administrated to cancerpatients to strengthen the capability of their immune system torecognize and kill the tumor cells. The main goal of a therapeuticcancer vaccine is to generate killer T cells (also called cytotoxic Tlymphocytes) specific for the tumor cells. To this end and to achieve apotent immune response, the vaccine must contain molecules calledantigens that are also present in the tumor and that need to bedelivered to Antigen Presenting Cells (APCs), especially dendritic cells(DCs), to allow cancer immunity to be initiated. The DCs process thesetumor antigens into small peptides that are presented on cell surfaceexpressed MHC class I or MHC class II molecules to T cells. Peptidesthat are then recognized by T cells and thereby induce their stimulationare called epitopes. Presentation by MHC class I and MHC class IImolecules allows activation of two classes of T cells, CD8+ cytotoxic Tlymphocytes (CTLs) and CD4+ helper T (T_(h)) cells, respectively. Inaddition, to become fully activated, beside antigen recognition T cellsrequire a second signal, the co-stimulatory signal, which is antigennon-specific and is provided by the interaction between co-stimulatorymolecules expressed on the surface of APCs and the T cell. Therefore twomajor requirements for an efficient therapeutic cancer vaccine are thespecificity of the tumor antigens and the ability to deliver themefficiently to DCs.

Taken together, induction of a tumor specific immune response thusrequires three main steps: (i) an antigen must be delivered to dendriticcells, which will process it into epitopes, (ii) dendritic cells shouldreceive a suitable activation signal, and (iii) activated tumorantigen-loaded dendritic cells must generate T-cell mediated immuneresponses in the lymphoid organs.

Since tumor cells can escape the immune system by down-regulatingexpression of individual antigens (passive immune escape),multi-epitopic antigen delivery provides an advantage. Indeed, proteinbased vaccines allow multi-epitopic antigen delivery to antigenpresenting cells (APCs) such as dendritic cells (DCs) without thelimitation of restriction to a single MHC allele. Another strength islong-lasting epitope presentation recently described in dendritic cellsloaded with proteins (van Montfoort N, Camps M G, Khan S, Filippov D V,Weterings J J, Griffith J M, et al. Antigen storage compartments inmature dendritic cells facilitate prolonged cytotoxic T lymphocytecross-priming capacity. Proceedings of the National Academy of Sciencesof the United States of America 2009;106(16):6730-5). Furthermore,proteins require uptake and processing by DCs to achieve MHC restrictedpresentation of their constituent epitopes. This reduces the risk ofinducing peripheral tolerance as has been shown after vaccination withshort peptides that do not have such stringent processing requirements(Toes R E, Offringa R, Blom R J, Melief C J, Kast W M. Peptidevaccination can lead to enhanced tumor growth through specific T-celltolerance induction. Proceedings of the National Academy of Sciences ofthe United States of America 1996;93(15):7855-60).

However, most soluble proteins are generally degraded in endolysosomesand are poorly cross-presented on MHC class I molecules and aretherefore poorly immunogenic for CD8+ T cell responses (Rosalia R A,Quakkelaar E D, Redeker A, Khan S, Camps M, Drijfhout J W, et al.Dendritic cells process synthetic long peptides better than wholeprotein, improving antigen presentation and T-cell activation. Europeanjournal of immunology 2013;43(10):2554-65). Moreover, although matureDCs are more potent than immature DCs in priming and eliciting T-cellresponses (Apetoh L, Locher C, Ghiringhelli F, Kroemer G, Zitvogel L.Harnessing dendritic cells in cancer. Semin Immunol. 2011; 23:42-49),they lose the ability to efficiently take up exogenous antigens,particularly for MHC class II restricted antigens (Banchereau J,Steinman R M. Dendritic cells and the control of immunity. Nature. 1998;392:245-252). As a result, peptide-pulsed DCs as vaccines have severallimitations. For example, peptide degradation, rapid MHC class Iturnover, and the disassociation of peptide from MHC class I moleculesduring the preparation and injection of DC/peptides may result in shorthalf-lives of MHC class I/peptide complexes on the DC surface, leadingto weak T-cell responses.

To improve the efficacy of protein-based vaccine delivery, the use ofcell penetrating peptides for intracellular delivery of cancer peptidesinto DCs has been proposed (Wang R F, Wang H Y. Enhancement of antitumorimmunity by prolonging antigen presentation on dendritic cells. NatBiotechnol. 2002; 20:149-156). Cell penetrating peptides (CPPs) arepeptides of 8 to 40 residues that have the ability to cross the cellmembrane and enter into most cell types (Copolovici D M, Langel K,Eriste E, Langel U. Cell-penetrating peptides: design, synthesis, andapplications. ACS nano 2014;8(3):1972-94, Milletti F. Cell-penetratingpeptides: classes, origin, and current landscape. Drug Discov Today2012). Alternatively, they are also called protein transduction domain(PTDs) reflecting their origin as occurring in natural proteins. Severalpotent CPPs have been identified from proteins, including the Tatprotein of human immunodeficiency virus, the VP22 protein of herpessimplex virus, and fibroblast growth factor (Berry C C. Intracellulardelivery of nanoparticles via the HIV-1 tat peptide. Nanomedicine. 2008;3:357-365; Deshayes S, Morris M C, Divita G, Heitz F. Cell-penetratingpeptides: Tools for intracellular delivery of therapeutics. Cell MolLife Sci. 2005; 62:1839-1849; Edenhofer F. Protein transductionrevisited: Novel insights into the mechanism underlying intracellulardelivery of proteins. Curr Pharm Des. 2008; 14:3628-3636; Gupta B,Levchenko T S, Torchilin V P. Intracellular delivery of large moleculesand small particles by cell-penetrating proteins and peptides. Adv DrugDeliv Rev. 2005; 57:637-651; Torchilin V P. Recent approaches tointracellular delivery of drugs and DNA and organelle targeting. AnnuRev Biomed Eng. 2006; 8:343-375). It was found that T-cell activityelicited by DC/TAT-TRP2 was 3- to 10-fold higher than that induced byDC/TRP2 (Wang H Y, Fu T, Wang G, Gang Z, Donna M P L, Yang J C, RestifoN P, Hwu P, Wang R F. Induction of CD4+ T cell-dependent antitumorimmunity by TAT-mediated tumor antigen delivery into dendritic cells. JClin Invest. 2002a; 109:1463-1470).

Moreover, subunits vaccines (peptides or proteins) are poorlyimmunogenic. Therefore in the context of therapeutic cancer vaccine, apotent adjuvant is mandatory to be added to the vaccine in order toincrease the level of co-stimulatory molecules on DCs and thereforeaugment the immune system's response to the target antigens. Adjuvantsaccomplish this task by mimicking conserved microbial components thatare naturally recognized by the immune system. They include,lipopolysaccharide (LPS), components of bacterial cell walls, andnucleic acids such as double-stranded RNA (dsRNA), single-stranded DNA(ssDNA), and unmethylated CpG dinucleotide-containing DNA. Theirpresence together with the vaccine can greatly increase the innateimmune response to the antigen. Furthermore, this adjuvant shouldpromote an adaptive immune response with CTLs and type polarized Th1rather than a humoral immune response resulting in antibody production.Different adjuvants have been evaluated, with a limited number havinggained regulatory approval for human use. These include Alum, MPL(monophosphoryl lipid A) and ASO₄ (Alum and MPL) in the US, and MF59(oil-in-water emulsion), ASO₄, liposomes in Europe (Lim, Y. T., Vaccineadjuvant materials for cancer immunotherapy and control of infectiousdisease. Clin Exp Vaccine Res, 2015. 4(1): p. 54-8).

Recently, Toll Like Receptor (TLR) ligands are emerging as promisingclass of adjuvants (Baxevanis, C. N., I. F. Voutsas, and O. E.Tsitsilonis, Toll-like receptor agonists: current status and futureperspective on their utility as adjuvants in improving anticancervaccination strategies. Immunotherapy, 2013. 5(5): p. 497-511). Asignificant development of cancer vaccine studies was thus to includevarious TLR agonists to vaccine formulations, including TLR-3 (polyI:C), TLR-4 (monophosphoryl lipid A; MPL), TLR-5 (flagellin), TLR-7(imiquimod), and TLR-9 (CpG) (Duthie M S, Windish H P, Fox C B, Reed SG. Use of defined TLR ligands as adjuvants within human vaccines.Immunol Rev. 2011; 239:178-196). The types of signaling and cytokinesproduced by immune cells after TLR stimulation control CD4+ T-celldifferentiation into Th1, Th2, Th17, and Treg cells. Stimulation ofimmune cells such as DCs and T cells by most TLR-based adjuvantsproduces proinflammatory cytokines and promotes Th1 and CD8+ T responses(Manicassamy S, Pulendran B. Modulation of adaptive immunity withToll-like receptors. Semin Immunol. 2009; 21:185-193).

Conjugating the vaccine to a TLR ligand is an attractive approach thatoffers several advantages over non-conjugated vaccines including (i)preferential uptake by the immune cells expressing the TLR, (ii) higherimmune response and (iii) reduced risk of inducing peripheral tolerance.Indeed, all the antigen presenting cells loaded with the antigen will besimultaneously activated. Different groups explored this approach withvarious TLR ligands being mainly linked chemically to the peptide orprotein vaccine (Zom G G, Khan S, Filippov D V, Ossendorp F. TLRligand-peptide conjugate vaccines: toward clinical application. AdvImmunol. 2012;114:177-201). As the chemical linkage to peptide is easilyperformed, the most highly investigated TLR ligands for conjugatevaccine are the TLR2 agonist Pam2Cys and Pam3Cys (Fujita, Y. and H.Taguchi, Overview and outlook of Toll-like receptor ligand-antigenconjugate vaccines. Ther Deliv, 2012. 3(6): p. 749-60).

However, to date the majority of cancer vaccines trials have shownlimited efficacy. One explanation is the lack of a therapy that cansimultaneously (i) stimulate multi-epitopic cytotoxic T cell-mediatedimmunity, (ii) induce T_(h) cells and (iii) promote immunologicalmemory. These three parameters are essential to generate potent, longlasting anti-tumor immunity. Indeed, CTLs specific for differentepitopes will allow destruction of more cancer cells within aheterogeneous tumor mass and avoid the outgrowth of antigen-lossvariants (tumor immune escape). T_(h) cells are involved in themaintenance of long-lasting cellular immunity and tumor infiltration byT_(h) cells is also an essential step for the recruitment and functionof CD8+ CTLs. Immunological memory is essential to protect against tumorrelapse.

In view of the above, it is the object of the present invention toovercome the drawbacks of current cancer vaccines outlined above and toprovide a novel complex for colorectal cancer immunotherapy applicationsrepresenting a more potent vaccine, having improved anti-tumor activityfor use in the prevention and/or treatment of colorectal cancer.

This object is achieved by means of the subject-matter set out below andin the appended claims.

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isnot intended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the term “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step but not the exclusion of any othernon-stated member, integer or step. The term “consist of” is aparticular embodiment of the term “comprise”, wherein any othernon-stated member, integer or step is excluded. In the context of thepresent invention, the term “comprise” encompasses the term “consistof”. The term “comprising” thus encompasses “including” as well as“consisting” e.g., a composition “comprising” X may consist exclusivelyof X or may include something additional e.g., X+Y.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the Invention.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means x±10%.

Complexes for Use According to the Present Invention

In a first aspect the present invention provides a complex comprising:

-   -   a) a cell penetrating peptide;    -   b) at least one antigen or antigenic epitope; and    -   c) at least one TLR peptide agonist,

wherein the components a)-c), i.e. the cell penetrating peptide, the atleast one antigen or antigenic epitope and the at least one TLR peptideagonist, are covalently linked, for use in the prevention and/ortreatment of colorectal cancer.

Such a complex for use according to the present invention providessimultaneous (i) stimulation of multi-epitopic cytotoxic T cell-mediatedimmunity, (ii) induction of T_(h) cells and (iii) promotion ofimmunological memory. Thereby, a complex for use according to thepresent invention provides a potent vaccine, in particular havingimproved anti-tumor activity.

Preferably, the complex for use according to the present invention is apolypeptide or a protein, in particular a recombinant polypeptide or arecombinant protein, preferably a recombinant fusion protein or arecombinant fusion polypeptide. The term “recombinant” as used hereinmeans that it (here: the polypeptide or the protein) does not occurnaturally. Accordingly, the complex for use according to the presentinvention, which is a recombinant polypeptide or a recombinant protein,typically comprises components a) to c), wherein components a) to c) areof different origins, i.e. do not naturally occur in this combination.

In the context of the present invention, i.e. throughout the presentapplication, the terms “peptide”, “polypeptide”, “protein” andvariations of these terms refer to peptide, oligopeptide, oligomer orprotein including fusion protein, respectively, comprising at least twoamino acids joined to each other preferably by a normal peptide bond,or, alternatively, by a modified peptide bond, such as for example inthe cases of isosteric peptides. A peptide, polypeptide or protein canbe composed of L-amino acids and/or D-amino acids. Preferably, apeptide, polypeptide or protein is either (entirely) composed of L-aminoacids or (entirely) of D-amino acids, thereby forming “retro-inversopeptide sequences”. The term “retro-inverso (peptide) sequences” refersto an isomer of a linear peptide sequence in which the direction of thesequence is reversed and the chirality of each amino acid residue isinverted (see e.g. Jameson et al., Nature, 368,744-746 (1994); Brady etal., Nature, 368,692-693 (1994)). In particular, the terms “peptide”,“polypeptide”, “protein also include “peptidomimetics” which are definedas peptide analogs containing non-peptidic structural elements, whichpeptides are capable of mimicking or antagonizing the biologicalaction(s) of a natural parent peptide. A peptidomimetic lacks classicalpeptide characteristics such as enzymatically scissile peptide bonds. Inparticular, a peptide, polypeptide or protein can comprise amino acidsother than the 20 amino acids defined by the genetic code in addition tothese amino acids, or it can be composed of amino acids other than the20 amino acids defined by the genetic code. In particular, a peptide,polypeptide or protein in the context of the present invention canequally be composed of amino acids modified by natural processes, suchas post-translational maturation processes or by chemical processes,which are well known to a person skilled in the art. Such modificationsare fully detailed in the literature. These modifications can appearanywhere in the polypeptide: in the peptide skeleton, in the amino acidchain or even at the carboxy- or amino-terminal ends. In particular, apeptide or polypeptide can be branched following an ubiquitination or becyclic with or without branching. This type of modification can be theresult of natural or synthetic post-translational processes that arewell known to a person skilled in the art. The terms “peptide”,“polypeptide”, “protein” in the context of the present invention inparticular also include modified peptides, polypeptides and proteins.For example, peptide, polypeptide or protein modifications can includeacetylation, acylation, ADP-ribosylation, amidation, covalent fixationof a nucleotide or of a nucleotide derivative, covalent fixation of alipid or of a lipidic derivative, the covalent fixation of aphosphatidylinositol, covalent or non-covalent cross-linking,cyclization, disulfide bond formation, demethylation, glycosylationincluding pegylation, hydroxylation, iodization, methylation,myristoylation, oxidation, proteolytic processes, phosphorylation,prenylation, racemization, seneloylation, sulfatation, amino acidaddition such as arginylation or ubiquitination. Such modifications arefully detailed in the literature (Proteins Structure and MolecularProperties (1993) 2nd Ed., T. E. Creighton, New York; Post-translationalCovalent Modifications of Proteins (1983) B. C. Johnson, Ed., AcademicPress, New York; Seifter et al. (1990) Analysis for proteinmodifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 andRattan et al., (1992) Protein Synthesis: Post-translationalModifications and Aging, Ann NY Acad Sci, 663: 48-62). Accordingly, theterms “peptide”, “polypeptide”, “protein” preferably include for examplelipopeptides, lipoproteins, glycopeptides, glycoproteins and the like.

However, in a particularly preferred embodiment, the complex for useaccording to the present invention is a “classical” peptide, polypeptideor protein, whereby a “classical” peptide, polypeptide or protein istypically composed of amino acids selected from the 20 amino acidsdefined by the genetic code, linked to each other by a normal peptidebond.

If the complex for use according to the present invention is apolypeptide or a protein, it is preferred that it comprises at least 50,at least 60, at least 70, preferably at least 80, at least 90, morepreferably at least 100, at least 110, even more preferably at least120, at least 130, particularly preferably at least 140, or mostpreferably at least 150 amino acid residues.

Component a)—Cell Penetrating Peptide

The CPP allows for efficient delivery, i.e. transport and loading, inparticular of at least one antigen or antigenic epitope, into theantigen presenting cells (APCs), in particular into the dendritic cells(DCs) and thus to the dendritic cells' antigen processing machinery.

The term “cell penetrating peptides” (“CPPs”) is generally used todesignate short peptides that are able to transport different types ofcargo molecules across plasma membrane, and, thus, facilitate cellularuptake of various molecular cargoes (from nanosize particles to smallchemical molecules and large fragments of DNA). “Cellularinternalization” of the cargo molecule linked to the cell penetratingpeptide generally means transport of the cargo molecule across theplasma membrane and thus entry of the cargo molecule into the cell.Depending on the particular case, the cargo molecule can, then, bereleased in the cytoplasm, directed to an intracellular organelle, orfurther presented at the cell surface. Cell penetrating ability, orinternalization, of the cell penetrating peptide or complex comprisingsaid cell penetrating peptide, according to the invention can be checkedby standard methods known to one skilled in the art, including flowcytometry or fluorescence microscopy of live and fixed cells,immunocytochemistry of cells transduced with said peptide or complex,and Western blot.

Cell penetrating peptides typically have an amino acid composition thateither contains a high relative abundance of positively charged aminoacids such as lysine or arginine or have a sequence that contains analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids. These two types of structures are referred toas polycationic or amphipathic, respectively. Cell-Penetrating peptidesare of different sizes, amino acid sequences, and charges but all CPPshave a common characteristic that is the ability to translocate theplasma membrane and facilitate the delivery of various molecular cargoesto the cytoplasm or to an organelle of a cell. At present, the theoriesof CPP translocation distinguish three main entry mechanisms: directpenetration in the membrane, endocytosis-mediated entry, andtranslocation through the formation of a transitory structure. CPPtransduction is an area of ongoing research. Cell-penetrating peptideshave found numerous applications in medicine as drug delivery agents inthe treatment of different diseases including cancer and virusinhibitors, as well as contrast agents for cell labeling and imaging.

Typically, cell penetrating peptides (CPPs) are peptides of 8 to 50residues that have the ability to cross the cell membrane and enter intomost cell types. Alternatively, they are also called proteintransduction domain (PTDs) reflecting their origin as occurring innatural proteins. Frankel and Pabo simultaneously to Green andLowenstein described the ability of the trans-activating transcriptionalactivator from the human immunodeficiency virus 1 (HIV-TAT) to penetrateinto cells (Frankel, A. D. and C. O. Pabo, Cellular uptake of the tatprotein from human immunodeficiency virus. Cell, 1988. 55(6): p.1189-93). In 1991, transduction into neural cells of the Antennapediahomeodomain (DNA-binding domain) from Drosophila melanogaster wasdescribed (Joliot, A., et al., Antennapedia homeobox peptide regulatesneural morphogenesis. Proc Natl Acad Sci U S A, 1991. 88(5): p. 1864-8).In 1994, the first 16-mer peptide CPP called Penetratin, having theamino acid sequence RQIKIYFQNRRMKWKK (SEQ ID NO: 1) was characterizedfrom the third helix of the homeodomain of Antennapedia (Derossi, D., etal., The third helix of the Antennapedia homeodomain translocatesthrough biological membranes. J Biol Chem, 1994. 269(14): p. 10444-50),followed in 1998 by the identification of the minimal domain of TAT,having the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2) required forprotein transduction (Vives, E., P. Brodin, and B. Lebleu, A truncatedHIV-1 Tat protein basic domain rapidly translocates through the plasmamembrane and accumulates in the cell nucleus. J Biol Chem, 1997.272(25): p. 16010-7). Over the past two decades, dozens of peptides weredescribed from different origins including viral proteins, e.g. VP22(Elliott, G. and P. O'Hare, Intercellular trafficking and proteindelivery by a herpesvirus structural protein. Cell, 1997. 88(2): p.223-33) and ZEBRA (Rothe, R., et al., Characterization of thecell-penetrating properties of the Epstein-Barr virus ZEBRAtrans-activator. J Biol Chem, 2010. 285(26): p. 20224-33), or fromvenoms, e.g. melittin (Dempsey, C. E., The actions of melittin onmembranes. Biochim Biophys Acta, 1990. 1031(2): p. 143-61), mastoporan(Konno, K., et al., Structure and biological activities of eumeninemastoparan-AF (EMP-AF), a new mast cell degranulating peptide in thevenom of the solitary wasp (Anterhynchium flavomarginatum micado).Toxicon, 2000. 38(11): p. 1505-15), maurocalcin (Esteve, E., et al.,Transduction of the scorpion toxin maurocalcine into cells. Evidencethat the toxin crosses the plasma membrane. J Biol Chem, 2005. 280(13):p. 12833-9), crotamine (Nascimento, F. D., et al., Crotamine mediatesgene delivery into cells through the binding to heparan sulfateproteoglycans. J Biol Chem, 2007. 282(29): p. 21349-60) or buforin(Kobayashi, S., et al., Membrane translocation mechanism of theantimicrobial peptide buforin 2. Biochemistry, 2004. 43(49): p.15610-6). Synthetic CPPs were also designed including the poly-arginine(R8, R9, R10 and R12) (Futaki, S., et al., Arginine-rich peptides. Anabundant source of membrane-permeable peptides having potential ascarriers for intracellular protein delivery. J Biol Chem, 2001. 276(8):p. 5836-40) or transportan (Pooga, M., et al., Cell penetration bytransportan. FASEB J, 1998. 12(1): p. 67-77). Any of the above describedCPPs may be used as cell penetrating peptide, i.e. as component a), inthe complex for use according to the present invention. In particular,the component a), i.e. the CPP, in the complex for use according to thepresent invention may comprise the minimal domain of TAT, having theamino acid sequence YGRKKRRQRRR (SEQ ID NO: 2). In particular, thecomponent a), i.e. the CPP, in the complex for use according to thepresent invention may comprise Penetratin having the amino acid sequenceRQIKIYFQNRRMKWKK (SEQ ID NO: 1).

Various CPPs, which can be used as cell penetrating peptide, i.e. ascomponent a), in the complex for use according to the present invention,are also disclosed in the review: Milletti, F., Cell-penetratingpeptides: classes, origin, and current landscape. Drug Discov Today 17(15-16): 850-60, 2012. In other words, the CPPs disclosed in Milletti,F., 2012, Cell-penetrating peptides: classes, origin, and currentlandscape. Drug Discov Today 17 (15-16): 850-60 can be used as cellpenetrating peptide, i.e. as component a), in the complex for useaccording to the present invention. This includes in particular cationicCPPs, amphipatic CPPs, and hydrophobic CPPs as well as CPPs derived fromheparan-, RNA- and DNA-binding proteins (cf. Table 1 of Milletti, F.,Cell-penetrating peptides: classes, origin, and current landscape. DrugDiscov Today 17 (15-16): 850-60, 2012), CPPs derived from signalpeptides (cf. Table 2 of Milletti, F., Cell-penetrating peptides:classes, origin, and current landscape. Drug Discov Today 17 (15-16):850-60, 2012), CPPs derived from antimicrobial peptides (cf. Table 3 ofMilletti, F., Cell-penetrating peptides: classes, origin, and currentlandscape. Drug Discov Today 17 (15-16): 850-60, 2012), CPPs derivedfrom viral proteins (cf. Table 4 of Milletti, F., Cell-penetratingpeptides: classes, origin, and current landscape. Drug Discov Today 17(15-16): 850-60, 2012), CPPs derived from various natural proteins (cf.Table 5 of Milletti, F., Cell-penetrating peptides: classes, origin, andcurrent landscape. Drug Discov Today 17 (15-16): 850-60, 2012), andDesigned CPPs and CPPs derived from peptide libraries (cf. Table 6 ofMilletti, F., Cell-penetrating peptides: classes, origin, and currentlandscape. Drug Discov Today 17 (15-16): 850-60, 2012).

Preferably, the cell penetrating peptide, which is comprised by thecomplex for use according to the present invention,

-   -   i) has a length of the amino acid sequence of said peptide of 5        to 50 amino acids in total, preferably of 10 to 45 amino acids        in total, more preferably of 15 to 45 amino acids in total;        and/or    -   ii) has an amino acid sequence comprising a fragment of the        minimal domain of ZEBRA, said minimal domain extending from        residue 170 to residue 220 of the ZEBRA amino acid sequence        according to SEQ ID NO: 3, wherein, optionally, 1, 2, 3, 4, or 5        amino acids have been substituted, deleted, and/or added without        abrogating said peptide's cell penetrating ability, or a        sequence variant of such a fragment.

Thereby, it is preferred that the cell penetrating peptide, which iscomprised by the complex for use according to the present invention,

-   -   i) has a length of the amino acid sequence of said peptide of 5        to 50 amino acids in total, preferably of 10 to 45 amino acids        in total, more preferably of 15 to 45 amino acids in total; and    -   ii) has an amino acid sequence comprising a fragment of the        minimal domain of ZEBRA, said minimal domain extending from        residue 170 to residue 220 of the ZEBRA amino acid sequence        according to SEQ ID NO: 3, wherein, optionally, 1, 2, 3, 4, or 5        amino acids have been substituted, deleted, and/or added without        abrogating said peptide's cell penetrating ability, or a        sequence variant of such a fragment.

Such preferred CPPs are disclosed in WO 2014/041505.

The term “ZEBRA” (also known as Zta, Z, EB1, or BZLF1) generally meansthe basic-leucine zipper (bZIP) transcriptional activator of theEpstein-Barr virus (EBV). The minimal domain of ZEBRA, which exhibitscell penetrating properties, has been identified as spanning fromresidue 170 to residue 220 of ZEBRA. The amino acid sequence of ZEBRA isdisclosed under NCBI accession number YP_401673 and comprises 245 aminoacids represented in SEQ ID NO: 3:

MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAYHVSTAPTGSWFSAPQPAPENAYQAYAAPQLFPVSDITQNQQTNQAGGEAPQPGDNSTVQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTRKPQQPESLEECDSELEIKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF(SEQ ID NO: 3-ZEBRA amino acid sequence (naturalsequence from Epstein-Barr virus (EBV))(YP_ 401673))

Recently, a CPP derived from the viral protein ZEBRA was described totransduce protein cargoes across biological membranes by both (i) directtranslocation and (ii) lipid raft-mediated endocytosis (Rothe R, LiguoriL, Villegas-Mendez A, Marques B, Grunwald D, Drouet E, et al.Characterization of the cell-penetrating properties of the Epstein-Barrvirus ZEBRA trans-activator. The Journal of biological chemistry2010;285(26):20224-33). The present inventors assume that these twomechanisms of entry should promote both MHC class I and II restrictedpresentation of cargo antigens to CD8+ and CD4+ T cells, respectively.Accordingly, such a CPP can deliver multi-epitopic peptides to dendriticcells (DCs), and subsequently to promote CTL and Th cell activation andanti-tumor function. Such a CPP can thus efficiently deliver the complexfor use according to the present invention to antigen presenting cells(APCs) and lead to multi-epitopic MHC class I and II restrictedpresentation.

In the context of the present invention, the term “MHC class I”designates one of the two primary classes of the MajorHistocompatibility Complex molecules. The MHC class I (also noted “MHCI”) molecules are found on every nucleated cell of the body. Thefunction of MHC class I is to display an epitope to cytotoxic cells(CTLs). In humans, MHC class I molecules consist of two polypeptidechains, α- and β2-microglobulin (b2m). Only the α chain is polymorphicand encoded by a HLA gene, while the b2m subunit is not polymorphic andencoded by the Beta-2 microglobulin gene. In the context of the presentinvention, the term “MHC class II” designates the other primary class ofthe Major Histocompatibility Complex molecules. The MHC class II (alsonoted “MHC II”) molecules are found only on a few specialized celltypes, including macrophages, dendritic cells and B cells, all of whichare dedicated antigen-presenting cells (APCs).

Preferably, the sequence variant of a fragment of the minimal domain ofZEBRA as described above shares, in particular over the whole length, atleast 70%, at least 75%, preferably at least 80%, more preferably atleast 85%, even more preferably at least 90%, particularly preferably atleast 95%, most preferably at least 99% amino acid sequence identitywith the fragment of the minimal domain of ZEBRA as described abovewithout abrogating the cell penetrating ability of the cell penetratingpeptide. In particular, a “fragment” of the minimal domain of ZEBRA asdefined above is preferably to be understood as a truncated sequencethereof, i.e. an amino acid sequence, which is N-terminally,C-terminally and/or intrasequentially truncated compared to the aminoacid sequence of the native sequence. Moreover, such a “fragment” of theminimal domain of ZEBRA has preferably a length of 5 to 50 amino acidsin total, preferably of 10 to 45 amino acids in total, more preferablyof 15 to 45 amino acids in total.

Accordingly, the term “sequence variant” as used in the context of thepresent invention, i.e. throughout the present application, refers toany alteration in a reference sequence. The term “sequence variant”includes nucleotide sequence variants and amino acid sequence variants.Preferably, a reference sequence is any of the sequences listed in the“Table of Sequences and SEQ ID Numbers” (Sequence listing), i.e. SEQ IDNO: 1 to SEQ ID NO: 70. Preferably, a sequence variant shares, inparticular over the whole length of the sequence, at least 70%, at least75%, preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, particularly preferably at least 95%, mostpreferably at least 99% sequence identity with a reference sequence,whereby sequence identity is calculated as described below. Inparticular, a sequence variant preserves the specific function of thereference sequence. Sequence identity is calculated as described below.In particular, an amino acid sequence variant has an altered sequence inwhich one or more of the amino acids in the reference sequence isdeleted or substituted, or one or more amino acids are inserted into thesequence of the reference amino acid sequence. As a result of thealterations, the amino acid sequence variant has an amino acid sequencewhich is at least 70%, at least 75%, preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, particularlypreferably at least 95%, most preferably at least 99% identical to thereference sequence. For example, variant sequences which are at least90% identical have no more than 10 alterations, i.e. any combination ofdeletions, insertions or substitutions, per 100 amino acids of thereference sequence.

In the context of the present invention, an amino acid sequence “sharinga sequence identity” of at least, for example, 95% to a query amino acidsequence of the present invention, is intended to mean that the sequenceof the subject amino acid sequence is identical to the query sequenceexcept that the subject amino acid sequence may include up to five aminoacid alterations per each 100 amino acids of the query amino acidsequence. In other words, to obtain an amino acid sequence having asequence of at least 95% identity to a query amino acid sequence, up to5% (5 of 100) of the amino acid residues in the subject sequence may beinserted or substituted with another amino acid or deleted, preferablywithin the above definitions of variants or fragments. The same, ofcourse, also applies similarly to nucleic acid sequences.

For (amino acid or nucleic acid) sequences without exact correspondence,a “% identity” of a first sequence may be determined with respect to asecond sequence. In general, these two sequences to be compared arealigned to give a maximum correlation between the sequences. This mayinclude inserting “gaps” in either one or both sequences, to enhance thedegree of alignment. A % identity may then be determined over the wholelength of each of the sequences being compared (so-called globalalignment), that is particularly suitable for sequences of the same orsimilar length, or over shorter, defined lengths (so-called localalignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequencesare well known in the art. The percentage to which two sequences areidentical can e.g. be determined using a mathematical algorithm. Apreferred, but not limiting, example of a mathematical algorithm whichcan be used is the algorithm of Karlin et al. (1993), PNAS USA,90:5873-5877. Such an algorithm is integrated in the BLAST family ofprograms, e.g. BLAST or NBLAST program (see also Altschul et al., 1990,J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res,25:3389-3402), accessible through the home page of the NCBI at worldwide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1990), MethodsEnzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci.U.S.A 85, 2444-2448.). Sequences which are identical to other sequencesto a certain extent can be identified by these programmes. Furthermore,programs available in the Wisconsin Sequence Analysis Package, version9.1 (Devereux et al., 1984, Nucleic Acids Res., 387-395), for examplethe programs BESTFIT and GAP, may be used to determine the % identitybetween two polynucleotides and the % identity and the % homology oridentity between two polypeptide sequences. BESTFIT uses the “localhomology” algorithm of (Smith and Waterman (1981), J. Mol. Biol. 147,195-197.) and finds the best single region of similarity between twosequences.

More preferably, the fragments of the cell penetrating peptide accordingto the invention or the variants thereof as described above furtherretain said peptide's ability to present a cargo molecule such asantigens or antigenic epitopes at the surface of a cell, such as anantigen-presenting cell, in the context of MHC class I and/or MHC classII molecules. The ability of a cell penetrating peptide or complexcomprising said cell penetrating peptide to present a cargo moleculesuch as antigens or antigenic epitopes at the surface of a cell in thecontext of MHC class I and/or MHC class II molecules can be checked bystandard methods known to one skilled in the art, including capacity tostimulate proliferation and/or function of MHC-restricted CD4+ or CD8+ Tcells with specificity for these epitopes.

The preferred cell penetrating peptide, which

-   -   i) has a length of the amino acid sequence of said peptide of 5        to 50 amino acids in total, preferably of 10 to 45 amino acids        in total, more preferably of 15 to 45 amino acids in total;        and/or    -   ii) has an amino acid sequence comprising a fragment of the        minimal domain of ZEBRA, said minimal domain extending from        residue 170 to residue 220 of the ZEBRA amino acid sequence        according to SEQ ID NO: 3, wherein, optionally, 1, 2, 3, 4, or 5        amino acids have been substituted, deleted, and/or added without        abrogating said peptide's cell penetrating ability, or a variant        of such a fragment

preferably comprises an amino acid sequence having at least oneconservatively substituted amino acid compared to the referencedsequence, meaning that a given amino acid residue is replaced by aresidue having similar physiochemical characteristics.

Generally, substitutions for one or more amino acids present in thereferenced amino acid sequence should be made conservatively. Examplesof conservative substitutions include substitution of one aliphaticresidue for another, such as Ile, VaI, Leu, or Ala for one another, orsubstitutions of one polar residue for another, such as between Lys andArg; Glu and Asp; or Gln and Asn. Other such conservative substitutions,for example, substitutions of entire regions having similarhydrophobicity properties, are well known (Kyte and Doolittle, 1982, J.Mol. Biol. 157(1):105-132). Substitutions of one or more L-amino acidswith one or more D-amino acids are to be considered as conservativesubstitutions in the context of the present invention. Exemplary aminoacid substitutions are presented in Table 1 below:

TABLE 1 Original residues Examples of substitutions Ala (A) Val, Leu,Ile, Gly Arg (R) His, Lys Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q)Asn Glu (E) Asp Gly (G) Pro, Ala His (H) Lys, Arg Ile (I) Leu, Val, Met,Ala, Phe Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, His Met (M) Leu,Ile, Phe Phe (F) Leu, Val, Ile, Tyr, Trp, Met Pro (P) Ala, Gly Ser (S)Thr Thr (T) Ser Trp (W) Tyr, Phe Tyr (Y) Trp, Phe Val (V) Ile, Met, Leu,Phe, Ala

Particularly preferably, the preferred cell penetrating peptide, which

-   -   i) has a length of the amino acid sequence of said peptide of 5        to 50 amino acids in total, preferably of 10 to 45 amino acids        in total, more preferably of 15 to 45 amino acids in total;        and/or    -   ii) has an amino acid sequence comprising a fragment of the        minimal domain of ZEBRA, said minimal domain extending from        residue 170 to residue 220 of the ZEBRA amino acid sequence        according to SEQ ID NO: 3, wherein, optionally, 1, 2, 3, 4, or 5        amino acids have been substituted, deleted, and/or added without        abrogating said peptide's cell penetrating ability, or a variant        of such a fragment

comprises a Cys (C) substituted into a Ser (S), at the equivalent ofposition 189 relative to ZEBRA amino acid sequence of SEQ ID NO: 3.

Thereby, it is preferred that such a preferred cell penetrating peptidehas an amino acid sequence comprising a sequence according to thefollowing general formula (I):

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁SX₁₃X₁₄X₁₅X₁₆X₁₇

with 0, 1, 2, 3, 4, or 5 amino acids which are substituted, deleted,and/or added without abrogating said peptide's cell penetrating ability,wherein

-   -   X₁ is K, R, or H, preferably X₁ is K or R;    -   X₂ is R, K, or H, preferably X₂ is R or K;    -   X₃ is Y, W, or F, preferably X₃ is Y, W, or F;    -   X₄ is K, R, or H, preferably X₄ is K or R;    -   X₅ is N or Q;    -   X₆ is R, K, or H, preferably X₆ is R or K;    -   X₇ is V, I, M, L, F, or A, preferably X₇ is V, I, M or L;    -   X₈ is A, V, L, I, or G, preferably X₈ is A or G;    -   X₉ is S or T;    -   X₁₀ is R, K, or H, preferably X₁₀ is R or K;    -   X₁₁ is K, R, or H, preferably X₁₁ is K or R;    -   X₁₃ is R, K, or H, preferably X₁₃ is R or K;    -   X₁₄ is A, V, L, I, or G, preferably X₁₄ is A or G;    -   X₁₅ is K, R, or H, preferably X₁₅ is K or R;    -   X₁₆ is F, L, V, I, Y, W, or M, preferably X₁₆ is F, Y or W; and    -   X₁₇ is K, R, or H, preferably X₁₇ is K or R.

Preferably, such a peptide, polypeptide or protein is either (entirely)composed of L-amino acids or (entirely) of D-amino acids, therebyforming “retro-inverso peptide sequences”. The term “retro-inverso(peptide) sequences” refers to an isomer of a linear peptide sequence inwhich the direction of the sequence is reversed and the chirality ofeach amino acid residue is inverted (see e.g. Jameson et al., Nature,368,744-746 (1994); Brady et al., Nature, 368,692-693 (1994)).

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₂ is R.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₃ is Y.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₄ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₅ is N.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₆ is R.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₇ is V.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₈ is A.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₉ is S.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₀ is R.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₁ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₃ is R.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₄ is A.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₅ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₆ is F.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₇ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein the amino acid at position equivalent to position 12 relative togeneral formula (I) is a Ser (S).

It is also particularly preferred, that the preferred cell penetratingpeptide, which

-   -   i) has a length of the amino acid sequence of said peptide of 5        to 50 amino acids in total, preferably of 10 to 45 amino acids        in total, more preferably of 15 to 45 amino acids in total;        and/or    -   ii) has an amino acid sequence comprising a fragment of the        minimal domain of ZEBRA, said minimal domain extending from        residue 170 to residue 220 of the ZEBRA amino acid sequence        according to SEQ ID NO: 3, wherein, optionally, 1, 2, 3, 4, or 5        amino acids have been substituted, deleted, and/or added without        abrogating said peptide's cell penetrating ability, or a variant        of such a fragment

comprises or consists of an amino acid sequence selected from the groupconsisting of amino acid sequences according to SEQ ID NO: 4-13, orsequence variants thereof without abrogating said peptide's cellpenetrating ability, preferably sequence variants having 0, 1, 2, 3, 4,or 5 amino acids substituted, deleted and/or added without abrogatingsaid peptide's cell penetrating ability.

CPP1 (Z11): (SEQ ID NO: 4) KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCCPP2 (Z12): (SEQ ID NO: 5) KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKCPP3 (Z13): (SEQ ID NO: 6) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKCPP4 (Z14): (SEQ ID NO: 7) KRYKNRVASRKSRAKFKQLLQHYREVAAAK CPP5 (Z15):(SEQ ID NO: 8) KRYKNRVASRKSRAKFK CPP6 (Z16): (SEQ ID NO: 9)QHYREVAAAKSSEND CPP7 (Z17): (SEQ ID NO: 10) QLLQHYREVAAAK CPP8 (Z18):(SEQ ID NO: 11) REVAAAKSSENDRLRLLLK CPP9 (Z19): (SEQ ID NO: 12) KRYKNRVACPP10 (Z20): (SEQ ID NO: 13) VASRKSRAKFK

Thereby, a cell penetrating peptide is particularly preferred, which hasan amino acid sequence comprising or consisting of an amino acidsequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14),SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18), or sequencevariants thereof without abrogating said peptide's cell penetratingability, preferably sequence variants having 0, 1, 2, 3, 4, or 5 aminoacids substituted, deleted and/or added without abrogating saidpeptide's cell penetrating ability. Moreover, a cell penetrating peptideis more preferred, which has an amino acid sequence comprising orconsisting of an amino acid sequence according to SEQ ID NO: 6(CPP3/Z13) or SEQ ID NO: 7 (CPP4/Z14) or sequence variants thereofwithout abrogating said peptide's cell penetrating ability, preferablysequence variants having 0, 1, 2, 3, 4, or 5 amino acids substituted,deleted and/or added without abrogating said peptide's cell penetratingability. Moreover, a cell penetrating peptide is most preferred, whichhas an amino acid sequence comprising or consisting of an amino acidsequence according to SEQ ID NO: 6 (CPP3/Z13) or sequence variantsthereof without abrogating said peptide's cell penetrating ability,preferably sequence variants having 0, 1, 2, 3, 4, or 5 amino acidssubstituted, deleted and/or added without abrogating said peptide's cellpenetrating ability.

In one preferred embodiment, the cell penetrating peptide according tothe invention has an amino acid sequence comprising or consisting of SEQID NO: 6 (CPP3/Z13).

In another preferred embodiment, the cell penetrating peptide accordingto the invention has an amino acid sequence comprising or consisting ofSEQ ID NO: 7 (CPP4/Z14).

In another preferred embodiment, the cell penetrating peptide accordingto the invention has an amino acid sequence comprising or consisting ofSEQ ID NO: 8 (CPP5/Z15).

In another preferred embodiment, the cell penetrating peptide accordingto the invention has an amino acid sequence comprising or consisting ofSEQ ID NO: 11 (CPP8/Z18).

It will be understood by one skilled in the art that the primary aminoacid sequence of the cell penetrating peptide of the invention mayfurther be post-translationally modified, such as by glycosylation orphosphorylation, without departing from the invention.

In a further embodiment, the cell penetrating peptide according to theinvention optionally further comprises, in addition to its amino acidsequence as described above, any one of, or any combination of:

-   -   (i) a nuclear localization signal (NLS). Such signals are well        known to the skilled person and are described in Nair et al.        (2003, Nucleic Acids Res. 31(1: 397-399)    -   (ii) a targeting peptide, including tumor homing peptides such        as those described in Kapoor et al. (2012, PLoS ONE 7(4):        e35187) and listed in        crdd.osdd.net/raghava/tumorhope/general.php?

Preferably, the cell penetrating peptide according to the invention islinked to an antigen or antigenic epitope and facilitates the cellularinternalization of said antigen or antigenic epitope.

The complex for use according to the present invention may comprise onesingle cell penetrating peptide or more than one cell penetratingpeptides. Preferably, the complex for use according to the presentinvention comprises no more than five cell penetrating peptides, morepreferably the complex for use according to the present inventioncomprises no more than four cell penetrating peptides, even morepreferably the complex for use according to the present inventioncomprises no more than three cell penetrating peptides, particularlypreferably the complex for use according to the present inventioncomprises no more than two cell penetrating peptides and most preferablythe complex for use according to the present invention comprises onesingle cell penetrating peptide.

Component b)—Antigen/Antigenic Epitope

The complex for use according to the present invention comprises ascomponent b) at least one antigen or antigenic epitope.

As used herein, an “antigen” is any structural substance which serves asa target for the receptors of an adaptive immune response, in particularas a target for antibodies, T cell receptors, and/or B cell receptors.An “epitope”, also known as “antigenic determinant”, is the part (orfragment) of an antigen that is recognized by the immune system, inparticular by antibodies, T cell receptors, and/or B cell receptors.Thus, one antigen has at least one epitope, i.e. a single antigen hasone or more epitopes. In the context of the present invention, the term“epitope” is mainly used to designate T cell epitopes, which arepresented on the surface of an antigen-presenting cell, where they arebound to Major Histocompatibility Complex (MHC). T cell epitopespresented by MHC class I molecules are typically, but not exclusively,peptides between 8 and 11 amino acids in length, whereas MHC class IImolecules present longer peptides, generally, but not exclusively,between 12 and 25 amino acids in length.

Preferably, in the complex for use according to the present invention,the at least one antigen or antigenic epitope is selected from the groupconsisting of (i) a peptide, a polypeptide, or a protein, (ii) apolysaccharide, (iii) a lipid, (iv) a lipoprotein or a lipopeptide, (v)a glycolipid, (vi) a nucleic acid, and (vii) a small molecule drug or atoxin. Thus, the at least one antigen or antigenic epitope may be apeptide, a protein, a polysaccharide, a lipid, a combination thereofincluding lipoproteins and glycolipids, a nucleic acid (e.g. DNA, siRNA,shRNA, antisense oligonucleotides, decoy DNA, plasmid), or a smallmolecule drug (e.g. cyclosporine A, paclitaxel, doxorubicin,methotrexate, 5-aminolevulinic acid), or any combination thereof inparticular if more than one antigen or antigenic epitope is comprised bythe inventive complex.

It is understood that the at least one antigen or antigenic epitope cancomprise for example at least one, i.e. one or more, peptides,polypeptides or proteins linked together and/or at least one, i.e. oneor more, nucleic acids, e.g. where each one encodes one peptide orpolypeptide. Also the at least one antigen or antigenic epitope can be acombination of a protein, a lipid, and/or a polysaccharide includinglipoproteins and glycolipids. Thus, in particular if the complex for useaccording to the present invention comprises more than one antigen orantigenic epitope, it can comprise more than one peptide, polypeptide,or protein, more than one polysaccharide, more than one lipid, more thanone lipoprotein, more than one glycolipid, more than one nucleic acid,more than one small molecule drug or toxin, or a combination thereof.

Preferably, the complex for use according to the invention comprises atleast one antigen or antigenic epitope comprising one or more epitope(s)from a cancer/tumor-associated antigen, a cancer/tumor-specific antigen,and/or an antigenic protein from a pathogen, including viral, bacterial,fungal, protozoal and multicellular parasitic antigenic protein.

More preferably, the at least one antigen or antigenic epitope comprisesor consists of (i) at least one pathogen epitope and/or (ii) at leastone cancer/tumor epitope, in particular at least one tumor epitope. Mostpreferably, the at least one antigen or antigenic epitope comprises orconsists of at least one cancer/tumor epitope, in particular at leastone tumor epitope.

It is particularly preferred that the complex for use according to thepresent invention comprises only such antigen(s) or antigenicepitope(s), which are cancer/tumor-associated antigen(s),cancer/tumor-specific antigen(s) and/or cancer/tumor epitope(s); inparticular, which are tumor-associated antigen(s), tumor-specificantigen(s), and/or tumor epitope(s).

As used herein, “cancer epitope” means an epitope from acancer-associated antigen or from a cancer-specific antigen.Accordingly, “tumor epitope” means an epitope from a tumor-associatedantigen or from a tumor-specific antigen. Such epitopes are typicallyspecific (or associated) for a certain kind of cancer/tumor. Inparticular, cancer/tumor-associated (also cancer/tumor-related) antigensare antigens, which are expressed by both, cancer/tumor cells and normalcells.

Accordingly, those antigens are normally present since birth (or evenbefore). Accordingly, there is a chance that the immune system developedself-tolerance to those antigens. Cancer/tumor-specific antigens, incontrast, are antigens, which are expressed specifically by cancer/tumorcells, but not by normal cells. Cancer/tumor-specific antigens includein particular neoantigens. In general neoantigens are antigens, whichwere not present before and are, thus, “new” to the immune system.Neoantigens are typically due to somatic mutations. In the context ofcancer/tumors, cancer/tumor-specific neoantigens were typically notpresent before the cancer/tumor developed and cancer/tumor-specificneoantigens are usually encoded by somatic gene mutations in thecancerous cells/tumor cells. Since neoantigens are new to the immunesystem, the risk of self-tolerance of those antigens is considerablylower as compared to cancer/tumor-associated antigens. However, everycancer's set of tumor-specific mutations appears to be unique.Accordingly, in the context of the present invention it is preferredthat such cancer/tumor-specific antigens, in particular neoantigens, areidentified in a subject diagnosed with colorectal cancer by methodsknown to the skilled person, e.g., cancer genome sequencing. Afteridentification, the respective cancer/tumor-specific neoantigens and/orcancer/tumor-specific neoantigenic epitopes are used in a complex foruse according to the present invention.

Preferably, a complex for use according to the present inventioncomprises one or more cancer/tumor-associated epitopes and/or one ormore cancer/tumor-associated antigens (but preferably nocancer/tumor-specific epitopes). It is also preferred that a complex foruse according to the present invention comprises one or morecancer/tumor-specific epitopes and/or one or more cancer/tumor-specificantigens (but preferably no cancer/tumor-associated epitopes). A complexfor use according to the present invention may also preferably compriseboth, (i) one or more cancer/tumor-associated epitopes and/or one ormore cancer/tumor-associated antigens and (ii) one or morecancer/tumor-specific epitopes and/or one or more cancer/tumor-specificantigens.

In particular, the cancer/tumor with which the antigens or antigenicepitopes are associated or for which the antigens or antigenic epitopesare specific is colorectal cancer as described herein. Thus, theantigens are preferably CRC-associated or CRC-specific antigens and theepitopes are preferably CRC-associated or CRC-specific epitopes.

Suitable cancer/tumor epitopes can be retrieved for example fromcancer/tumor epitope databases, e.g. from van der Bruggen P, StroobantV, Vigneron N, Van den Eynde B. Peptide database: T cell-defined tumorantigens. Cancer Immun 2013; URL: www.cancerimmunity.org/peptide/,wherein human tumor antigens recognized by CD4+ or CD8+ T cells areclassified into four major groups on the basis of their expressionpattern, or from the database “Tantigen” (TANTIGEN version 1.0, Dec. 1,2009; developed by Bioinformatics Core at Cancer Vaccine Center,Dana-Farber Cancer Institute; URL: cvc.dfci.harvard.edu/tadb/). Examplesof cancer/tumor epitopes include e.g. TRP2-derived epitopes,glycoprotein 100 (gp100) melanoma antigen-derived epitopes, glycoprotein70 (gp70) antigen-derived epitopes, survivin epitopes, IEa epitopes,IL13rα2, Epha2 (ephrin type-A receptor 2), immunogenic fragmentsthereof, and fusions of such antigens and/or fragments. Furthermore,examples of cancer/tumor epitopes include epitopes of neoantigens, suchas, for example, a neoantigen from MC-38 tumor cell line as described byYadav et al. Nature. 2014 Nov 27;515(7528):572-6. As described above,neoantigens are antigens, which are entirely absent from the normalhuman genome. As compared with nonmutated self-antigens, neoantigens areof relevance to tumor control, as the quality of the T cell pool that isavailable for these antigens is not affected by central T celltolerance. In particular, neoantigens may be based on individual tumorgenomes. Potential neoantigens may be predicted by methods known to theskilled person, such as cancer genome sequencing or deep-sequencingtechnologies identifying mutations within the protein-coding part of the(cancer) genome.

Specific examples of cancer/tumor-associated, in particulartumor-related, or tissue-specific antigens useful in a complex for useaccording to the present invention include, but are not limited to, thefollowing antigens: Her-2/neu, SPAS-1, TRP-2, tyrosinase, MelanA/Mart-1, gplOO, BAGE, GAGE, GM2 ganglioside, kinesin 2, TATA elementmodulatory factor 1, tumor protein D52, MAGE D, ING2, HIP-55, TGF-1anti-apoptotic factor, HOM-Mel-40/SSX2, epithelial antigen (LEA 135),DF31MUC1 antigen (Apostolopoulos et al., 1996 Immunol. Cell. Biol. 74:457-464; Pandey et al., 1995, Cancer Res. 55: 4000-4003), MAGE-1,HOM-Mel-40/SSX2, NY-ESO-1, EGFR, CEA, Epha2, Epha4, PCDGF, HAAH,Mesothelin; EPCAM; NY-ESO-1, glycoprotein MUC1 and NIUC10 mucins p5(especially mutated versions), EGFR, cancer-associated serum antigen(CASA) and cancer antigen 125 (CA 125) (Kierkegaard et al., 1995,Gynecol. Oncol. 59: 251-254), the epithelial glycoprotein 40 (EGP40)(Kievit et al., 1997, Int. J. Cancer 71: 237-245), squamous cellcarcinoma antigen (SCC) (Lozza et al., 1997 Anticancer Res. 17:525-529), cathepsin E (Mota et al., 1997, Am. J Pathol. 150: 1223-1229),tyrosinase in melanoma (Fishman et al., 1997 Cancer 79: 1461-1464), cellnuclear antigen (PCNA) of cerebral cavernomas (Notelet et al., 1997Surg. Neurol. 47: 364-370), a 35 kD tumor-associated autoantigen inpapillary thyroid carcinoma (Lucas et al., 1996 Anticancer Res. 16:2493-2496), CDC27 (including the mutated form of the protein), antigenstriosephosphate isomerase, 707-AP, A60 mycobacterial antigen (Macs etal., 1996, J. Cancer Res. Clin. Oncol. 122: 296-300), Annexin II, AFP,ART-4, BAGE, β-catenin/m, BCL-2, bcr-abl, bcr-abl p190, bcr-abl p210,BRCA-1, BRCA-2, CA 19-9 (Tolliver and O'Brien, 1997, South Med. J. 90:89-90; Tsuruta at al., 1997 Urol. Int. 58: 20-24), CAMEL, CAP-1, CASP-8,CDC27/m, CDK-4/m, CEA (Huang et al., Exper Rev. Vaccines (2002)1:49-63),CT9, CT10, Cyp-B, Dek-cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1), EphA2(Zantek et al., Cell Growth Differ. (1999) 10:629-38; Carles-Kinch etal., Cancer Res. (2002) 62:2840-7), EphA4 (Cheng at al., 2002, CytokineGrowth Factor Rev. 13:75-85), tumor associated Thomsen-Friedenreichantigen (Dahlenborg et al., 1997, Int. J Cancer 70: 63-71), ELF2M,ETV6-AML1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7B, GAGE-8, GnT-V, gp100 (Zajac et al., 1997, Int. J Cancer 71:491-496), HAGE, HER2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2,hTERT, hTRT, iCE, inhibitors of apoptosis (e.g., survivin), KH-1adenocarcinoma antigen (Deshpande and Danishefsky, 1997, Nature 387:164-166), KIAA0205, K-ras, LAGE, LAGE-1, LDLR/FUT, MAGE-1, MAGE-2,MAGE-3, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10,MAGE-A12, MAGE-B5, MAGE-B6, MAGE-C2, MAGE-C3, MAGE D, MART-1,MART-1/Melan-A (Kawakami and Rosenberg, 1997, Int. Rev. Immunol. 14:173-192), MC1R, MDM-2, Myosin/m, MUC1, MUC2, MUM-1, MUM-2, MUM-3,neo-polyA polymerase, NA88-A, NY-ESO-1, NY-ESO-1a (CAG-3), PAGE-4, PAP,Proteinase 3 (Molldrem et al., Blood (1996) 88:2450-7; Molldrem et al.,Blood (1997) 90:2529-34), P15, p190, Pm1/RARα, PRAME, PSA, PSM, PSMA,RAGE, RAS, RCAS1, RU1, RU2, SAGE, SART-1, SART-2, SART-3, SP17, SPAS-1,TEL/AML1, TPI/m, Tyrosinase, TARP, TRP-1 (gp75), TRP-2, TRP-2/INT2,WT-1, and alternatively translated NY-ESO-ORF2 and CAMEL proteins,derived from the NY-ESO-1 and LAGE-1 genes. Numerous other cancerantigens are well known in the art.

Preferably, the cancer/tumor antigen or the cancer/tumor epitope is arecombinant cancer/tumor antigen or a recombinant cancer/tumor epitope.Such a recombinant cancer/tumor antigen or a recombinant cancer/tumorepitope may be designed by introducing mutations that change (add,delete or substitute) particular amino acids in the overall amino acidsequence of the native cancer/tumor antigen or the native cancer/tumorepitope. The introduction of mutations does not alter the cancer/tumorantigen or the cancer/tumor epitope so much that it cannot beuniversally applied across a mammalian subject, and preferably a humanor dog subject, but changes it enough that the resulting amino acidsequence breaks tolerance or is considered a foreign antigen in order togenerate an immune response. Another manner may be creating a consensusrecombinant cancer/tumor antigen or cancer/tumor epitope that has atleast 85% and up to 99% amino acid sequence identity to its'corresponding native cancer/tumor antigen or native cancer/tumorepitope; preferably at least 90% and up to 98% sequence identity; morepreferably at least 93% and up to 98% sequence identity; or even morepreferably at least 95% and up to 98% sequence identity. In someinstances the recombinant cancer/tumor antigen or the recombinantcancer/tumor epitope has 95%, 96%, 97%, 98%, or 99% amino acid sequenceidentity to its' corresponding native cancer/tumor antigen orcancer/tumor epitope. The native cancer/tumor antigen is the antigennormally associated with the particular cancer or cancer tumor.Depending upon the cancer/tumor antigen, the consensus sequence of thecancer/tumor antigen can be across mammalian species or within subtypesof a species or across viral strains or serotypes. Some cancer/tumorantigen do not vary greatly from the wild type amino acid sequence ofthe cancer/tumor antigen. The aforementioned approaches can be combinedso that the final recombinant cancer/tumor antigen or cancer/tumorepitope has a percent similarity to native cancer antigen amino acidsequence as discussed above. Preferably, however, the amino acidsequence of an epitope of a cancer/tumor antigen as described herein isnot mutated and, thus, identical to the reference epitope sequence.

As used herein “pathogen epitope” means an epitope from an antigenicprotein, an antigenic polysaccharide, an antigenic lipid, an antigeniclipoprotein or an antigenic glycolipid from a pathogen includingviruses, bacteria, fungi, protozoa and multicellular parasites.Antigenic proteins, polysaccharides, lipids, lipoproteins or glycolipidsfrom pathogens include, herewith, proteins, polysaccharides, lipids,lipoproteins and glycolipids, respectively, from pathogens responsibleof diseases which can be a target for vaccination including, forinstance, Amoebiasis, Anthrax, Buruli Ulcer (Mycobacterium ulcerans),Caliciviruses associated diarrhoea, Campylobacter diarrhoea, CervicalCancer (Human papillomavirus), Chlamydia trachomatis associated genitaldiseases, Cholera, Crimean-Congo haemorrhagic fever, Dengue Fever,Diptheria, Ebola haemorrhagic fever, Enterotoxigenic Escherichia coli(ETEC) diarrhoea, Gastric Cancer (Helicobacter pylori), Gonorrhea, GroupA Streptococcus associated diseases, Group B Streptococcus associateddiseases, Haemophilus influenzae B pneumonia and invasive disease,Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E diarrhoea, Herpessimplex type 2 genital ulcers, HIV/AIDS, Hookworm Disease, Influenza,Japanese encephalitis, Lassa Fever, Leishmaniasis, Leptospirosi, Livercancer (Hepatitis B), Liver Cancer (Hepatitis C), Lyme Disease, Malaria,Marburg haemorrhagic fever, Measles, Mumps, Nasopharyngeal cancer(Epstein-Barr virus), Neisseria meningitidis Meningitis, Parainfluenzaassociated pneumonia, Pertussis, Plague, Poliomyelitis, Rabies,Respiratory syncytial virus (RSV) pneumonia, Rift Valley fever,Rotavirus diarrhoea, Rubella, Schistosomiasis, Severe Acute RespiratorySyndrome (SARS), Shigellosis, Smallpox, Staphylococcus aureus associateddiseases, Stomach Cancer (Helicobacter pylori), Streptococcus pneumoniaeand invasive disease, Tetanus, Tick-borne encephalitis, Trachoma,Tuberculosis, Tularaemia, Typhoid fever, West-Nile virus associateddisease, Yellow fever.

Preferably, the at least one antigen or antigenic epitope will bepresented at the cell surface in an MHC class I and/or MHC class IIcontext and/or in a CD1 context, whereby presentation at the cellsurface in an MHC class I and/or MHC class II context is preferred. Thephrase “epitope presentation in the MHC class I context” refers inparticular to a CD8+ epitope lying in the groove of a MHC class Imolecule at the surface of a cell. The phrase “epitope presentation inthe MHC class II context” refers in particular to a CD4+ epitope lyingin the groove of a MHC class II molecule at the surface of a cell. Thephrase “epitope presentation in the CD1 context” refers in particular toa lipidic epitope lying in the groove of a cluster of differentiation 1molecule at the surface of a cell.

Advantageously, the complex for use according to the invention comprisesa cell penetrating peptide and at least one antigen or antigenicepitope, and allows the transport and presentation of said epitopes atthe cell surface of antigen presenting cells in an MHC class I and MHCclass II context, and is, thus, useful in vaccination and immunotherapy.

Preferably, the complex for use according to the present inventioncomprises at least one antigen or antigenic epitope, which is at leastone CD4+ epitope and/or at least one CD8+ epitope.

The terms “CD4+ epitope” or “CD4+-restricted epitope”, as used herein,designate an epitope recognized by a CD4+ T cell, said epitope inparticular consisting of an antigen fragment lying in the groove of aMHC class II molecule. A single CD4+ epitope comprised in the complexfor use according to the present invention preferably consists of about12-25 amino acids. It can also consist of, for example, about 8-25 aminoacids or about 6-100 amino acids.

The terms “CD8+ epitope” or “CD8+-restricted epitope”, as used herein,designate an epitope recognized by a CD8+ T cell, said epitope inparticular consisting of an antigen fragment lying in the groove of aMHC class I molecule. A single CD8+ epitope comprised in the complex foruse according to the present invention preferably consists of about 8-11amino acids. It can also consist of, for example, about 8-15 amino acidsor about 6-100 amino acids.

Preferably, the at least one antigen can comprise or the at least oneantigenic epitope can consist of a CD4+ epitope and/or a CD8+ epitopecorresponding to antigenic determinant(s) of a cancer/tumor-associatedantigen, a cancer/tumor-specific antigen, or an antigenic protein from apathogen. More preferably, the at least one antigen can comprise or theat least one antigenic epitope can consist of a CD4+ epitope and/or aCD8+ epitope corresponding to antigenic determinant(s) of acancer/tumor-associated antigen or a cancer/tumor-specific antigen. Mostpreferably, the at least one antigen can comprise or the at least oneantigenic epitope can consist of a CD4+ epitope and/or a CD8+ epitopecorresponding to antigenic determinant(s) of a tumor-associated antigenor a tumor-specific antigen.

It is also preferred that the complex for use according to the presentinvention comprises at least two antigens or antigenic epitopes, whereinat least one antigen or antigenic epitope comprises or consists a CD4+epitope and at least one antigen or antigenic epitope comprises orconsists a CD8+ epitope. It is now established that T_(h) cells (CD4+)play a central role in the anti-tumor immune response both in DClicensing and in the recruitment and maintenance of CTLs (CD8+) at thetumor site. Therefore, a complex for use according to the presentinvention comprising at least two antigens or antigenic epitopes,wherein at least one antigen or antigenic epitope comprises or consistsof a CD4+ epitope and at least one antigen or antigenic epitopecomprises or consists a CD8+ epitope, provides an integrated immuneresponse allowing simultaneous priming of CTLs and T_(h) cells and isthus preferable to immunity against only one CD8+ epitope or only oneCD4+ epitope. For example, the complex for use according to the presentinvention may preferably comprise an Ealpha-CD4+ epitope and agp100-CD8+ epitope.

Preferably, the complex for use according to the present inventioncomprises at least two antigens or antigenic epitopes, wherein the atleast two antigens or antigenic epitopes comprise or consist of at leasttwo, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or more, CD4+ epitopes and/or at leasttwo, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or more, CD8+ epitopes. Thereby, theat least two antigens or antigenic epitopes are preferably differentantigens or antigenic epitopes, more preferably the at least twoantigens or antigenic epitopes are different from each other butrelating to the same kind of tumor. A multi-antigenic vaccine will (i)avoid outgrowth of antigen-loss variants, (ii) target different tumorcells within a heterogeneous tumor mass and (iii) circumventpatient-to-patient tumor variability. Thus, the complex for useaccording to the present invention particularly preferably comprises atleast four antigens or antigenic epitopes, in particular with at leasttwo CD8+ epitopes and at least two CD4+ epitopes. Such a complex for useaccording to the present invention induces multi-epitopic CD8 CTLs andCD4 T_(h) cells to function synergistically to counter tumor cells andpromote efficient anti-tumor immunity. T_(h) cells are also involved inthe maintenance of long-lasting cellular immunity that was monitoredafter vaccination. Such a complex for use according to the presentinvention induces polyclonal, multi-epitopic immune responses andpoly-functional CD8+ and CD4+ T cells, and thus efficacious anti-tumoractivity.

Preferably, the complex for use according to the present inventioncomprises at least two antigens or antigenic epitopes, more preferablythe complex for use according to the present invention comprises atleast three antigens or antigenic epitopes, even more preferably thecomplex for use according to the present invention comprises at leastfour antigens or antigenic epitopes, particularly preferably the complexfor use according to the present invention comprises at least fiveantigens or antigenic epitopes and most preferably the complex for useaccording to the present invention comprises at least six antigens orantigenic epitopes. The antigens or antigenic epitopes comprised by thecomplex for use according to the present invention may be the same ordifferent, preferably the antigens or antigenic epitopes comprised bythe complex for use according to the present invention are differentfrom each other. Preferably, the complex for use according to thepresent invention comprises at least one CD4+ epitope and at least oneCD8+ epitope.

Preferably, the complex for use according to the present inventioncomprises more than one CD4+ epitope, e.g. two or more CD4+ epitopesfrom the same antigen or from different antigens, and preferably no CD8+epitope. It is also preferred that the complex for use according to thepresent invention comprises more than one CD8+ epitope, e.g. two or moreCD8+ epitopes from the same antigen or from different antigens, andpreferably no CD4+ epitope. Most preferably, however, the complex foruse according to the present invention comprises (i) at least one CD4+epitope, e.g. two or more CD4+ epitopes from the same antigen or fromdifferent antigens, and (ii) at least one CD8+ epitope, e.g. two or moreCD8+ epitopes from the same antigen or from different antigens.

For example, the complex for use according to the present invention maypreferably comprise a gp100-CD8+ epitope, an Ealpha-CD4+ epitope, and afurther CD4+ epitope and a further CD8+ epitope. Even more preferably,the complex for use according to the present invention may comprise apolypeptide or protein comprising a gp100-CD8+ epitope and anEalpha-CD4+ epitope. For example, such a polypeptide or proteincomprised by the complex for use according to the present inventioncomprises or consists of an amino acid sequence according to SEQ ID NO:14 or sequence variants thereof as defined above:

SEQ ID NO: 14 ESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQD WLGVPRFAKFASFEAQGALA NIAVDKANLD VEQLESIINF EKLTEWTGS(MAD5-cargo comprising OVA-CD4+, gp100-CD8+,Ealpha-CD4+, and OVA-CD8+ epitopes)

For example, the complex for use according to the present invention mayalso comprise a gp70-CD8+ epitope and/or a gp70-CD4+ epitope. Inparticular, the complex for use according to the present invention maycomprise a polypeptide or protein comprising a gp70-CD8+ epitope and/ora gp70-CD4+ epitope. For example, such a polypeptide or proteincomprised by the complex for use according to the present inventioncomprises or consists of an amino acid sequence according to SEQ ID NO:43 or sequence variants thereof as defined above:

SEQ ID NO: 43 VTYHSPSYAYHQFERRAILNRLVQFIKDRI(Mad8-cargo comprising a gp70-CD8+ and a gp70-CD4+ epitope)

For example, the complex for use according to the present invention maypreferably comprise at least one survivinepitope, such as a survivinCD8+ epitope and/or a survivin CD4+ epitope. More preferably, thecomplex for use according to the present invention may comprise apolypeptide or protein comprising a survivin CD8+ epitope and/or asurvivin CD4+ epitope. More preferably, the complex for use according tothe present invention may comprise a polypeptide or protein comprisingmore than one survivin CD8+ epitope and/or more than one survivin CD4+epitope, such as two different survivin CD8+ epitopes. For example, sucha polypeptide or protein comprised by the complex for use according tothe present invention comprises or consists of an amino acid sequenceaccording to SEQ ID NO: 44 or sequence variants thereof as definedabove:

SEQ ID NO: 44 NYRIATFKNWPFLEDCAMEELTVSEFLKLDRQR(Mad11-cargo comprising survivin CD8+ epitope 1and survivin CD8+ epitope 2)

For example, the complex for use according to the present invention maypreferably comprise an epitope from a neoantigen. Even more preferably,the complex for use according to the present invention may comprise apolypeptide or protein comprising an epitope from a neoantigen, such asthe neoantigen from MC-38 tumor cell line identified by Yadav et al.Nature. 2014 Nov 27;515(7528):572-6. For example, such a polypeptide orprotein comprised by the complex for use according to the presentinvention comprises or consists of an amino acid sequence according toSEQ ID NO: 42 or sequence variants thereof as defined above:

SEQ ID NO: 42 HLELASMTNMELMSSIV(Mad9-cargo comprising the epitope from aneoantigen as described by Yadav et al. Nature.2014 November 27;515(7528):572-6).

For example, the complex for use according to the present invention maypreferably comprise more than one, e.g. two or three, epitopes fromneoantigens. Even more preferably, the complex for use according to thepresent invention may comprise a polypeptide or protein comprising morethan one, e.g. two or three, epitopes from neoantigens, such as theneoantigens from MC-38 tumor cell line identified by Yadav et al.Nature. 2014 Nov 27;515(7528):572-6. For example, such a polypeptide orprotein comprised by the complex for use according to the presentinvention comprises or consists of an amino acid sequence according toSEQ ID NO: 63 or sequence variants thereof as defined above:

SEQ ID NO: 63 LFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEG(Mad12-cargo comprising the epitope from aneoantigen as described by Yadav et al. Nature.2014 November 27;515(7528):572-6).

Preferably, the at least one antigen or antigenic epitope comprised bythe complex for use according to the present invention is a peptide,polypeptide, or a protein. Examples of antigen or antigenic epitope ofpeptidic, polypeptidic, or proteic nature useful in the invention,include cancer/tumor antigens or antigenic epitopes thereof, allergyantigens or antigenic epitopes thereof, auto-immune self-antigens orantigenic epitopes thereof, pathogenic antigens or antigenic epitopesthereof, and antigens or antigenic epitopes thereof from viruses,preferably from cytomegalovirus (CMV), orthopox variola virus, orthopoxalastrim virus, parapox ovis virus, molluscum contagiosum virus, herpessimplex virus 1, herpes simplex virus 2, herpes B virus, varicellazoster virus, pseudorabies virus, human cytomegaly virus, human herpesvirus 6, human herpes virus 7, Epstein-Barr virus, human herpes virus 8,hepatitis B virus, chikungunya virus, O'nyong'nyong virus, rubivirus,hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellowfever virus, louping ill virus, St. Louis encephalitis virus, Japan Bencephalitis virus, Powassan virus, FSME virus, SARS, SARS-associatedcorona virus, human corona virus 229E, human corona virus Oc43,Torovirus, human T cell lymphotropic virus type I, human T celllymphotropic virus type II, HIV (AIDS), i.e. human immunodeficiencyvirus type 1 or human immunodeficiency virus type 2, influenza virus,Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Juninvirus, Machupo virus, Borna disease virus, Bunyamwera virus, Californiaencephalitis virus, Rift Valley fever virus, sand fly fever virus,Toscana virus, Crimean-Congo haemorrhagic fever virus, Hazara virus,Khasan virus, Hantaan virus, Seoul virus, Prospect Hill virus, Puumalavirus, Dobrava Belgrade virus, Tula virus, sin nombre virus, LakeVictoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, IvoryCoast Ebola virus, influenza virus A, influenza virus B, influenzaviruses C, parainfluenza virus, malaria parasite (Plasmodium falciparum,Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodiumknowlesi), Marburg virus, measles virus, mumps virus, respiratorysyncytial virus, human metapneumovirus, vesicular stomatitis Indianavirus, rabies virus, Mokola virus, Duvenhage virus, European batlyssavirus 1+2, Australian bat lyssavirus, adenoviruses A-F, humanpapilloma viruses, condyloma virus 6, condyloma virus 11, polyomaviruses, adeno-associated virus 2, rotaviruses, orbiviruses, varicellaincluding varizella zoster, etc., or antigens or antigenic epitopes fromleishmania, typanosomes, amibes, bacteria, etc., or may be selected fromepitopes or from variants of the above antigens or antigenic epitopes.Preferably, epitopes as well as variants of antigens as defined aboveexhibit a sequence homology or identity of about 10%, in particular atleast 10%, about 20%, in particular at least 20%, about 30%, inparticular at least 30%, about 40%, in particular at least 40%, about50%, in particular at least 50%, about 60%, in particular at least 60%,about 70%, in particular at least 70%, about 80%, in particular at least80%, about 90% in particular at least 90%, at least 95% or at least 98%with one of the antigens or antigen sequences as shown or describedabove. In this context, the definition of epitopes and variantssimilarly applies as defined.

Examples of antigens or antigenic epitopes in the category of peptide,polypeptide or protein include a combination of multiple glioma epitopessuch as those described in Novellino et al. (2005, Cancer ImmunolImmunother, 54(3):187-207), Vigneron et al. (2013, Cancer Immun.13:15).However, a single complex for use according to the present invention mayalso comprise only a subset, i.e. one or more of all of said gliomaepitopes. In such a case preferably different complexes according to thepresent invention comprise different subsets of all of said gliomaepitopes, so that for example a vaccine according to the presentinvention comprising such different complexes according to the presentinvention comprises all of said glioma epitopes but distributed in thedifferent complexes.

Moreover, a complex for use according to the invention may also compriseat least one antigen or antigenic epitope, wherein said antigen orantigenic epitope is a polysaccharide, a lipid, a lipoprotein, and/or aglycolipid, in particular a polysaccharidic, lipidic, lipoproteic,and/or glycolipidic epitope, which can be, for example, pathogenepitopes as defined herewith.

In particular, the complex for use according to the invention maycomprise at least one antigen or antigenic epitope, wherein said antigenor antigenic epitope is polysaccharidic, lipidic, lipoproteic, and/orglycolipidic, including viral, bacterial, fungal, protozoal andmulticellular parasitic antigens or antigenic epitopes.

Preferably, said epitopes will be presented at the cell surface in anMHC class I and/or MHC class II context.

Preferably, said lipidic epitopes will be presented at the cell surfacein a CD1 (cluster of differentiation 1) context.

The complex for use according to the present invention may also compriseat least one antigen or antigenic epitope, wherein said antigen orantigenic epitope is a small molecule drug or toxin.

Examples of cargo molecules within the category of small molecule drugsor toxins useful in the invention include cyclosporine A, paclitaxel,doxorubicin, methotrexate, 5-aminolevulinic acid, diphtheria toxin,sunitinib and those molecules reviewed in De wit Amer (2010, NeuroOncol, 12(3):304-16).

The complex for use according to the present invention comprises atleast one antigen or antigenic epitope, preferably the complex for useaccording to the present invention comprises more than one antigen orantigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreantigens or antigenic epitopes, more preferably the complex for useaccording to the present invention comprises (at least) two or threeantigens or antigenic epitopes, even more preferably the complex for useaccording to the present invention comprises (at least) four or fiveantigens or antigenic epitopes.

If more than one antigen or antigenic epitope is comprised by thecomplex for use according to the present invention it is understood thatsaid antigen or antigenic epitope is in particular also covalentlylinked in the complex for use according to the present invention, e.g.to another antigen or antigenic epitope and/or to a component a), i.e. acell penetrating peptide, and/or to a component c), i.e. a TLR peptideagonist.

The various antigens or antigenic epitopes comprised by the complex foruse according to the present invention may be the same or different.Preferably, the various antigens or antigenic epitopes comprised by thecomplex for use according to the present invention are different fromeach other, thus providing a multi-antigenic and/or multi-epitopiccomplex.

Moreover, it is preferred that the more than one antigen or antigenicepitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens orantigenic epitopes, are positioned consecutively in the complex for useaccording to the present invention. This means in particular that allantigens and/or antigenic epitopes comprised by the complex arepositioned in a stretch, which is neither interrupted by component a),i.e. a cell penetrating peptide, nor by component c), i.e. a TLR peptideagonist. Rather, component a) and component c) are positioned in thecomplex for example before or after such a stretch of all antigensand/or antigenic epitopes. However, the antigens and/or antigenicepitopes positioned consecutively in such a way may be linked to eachother for example by a spacer or linker as described below, which isneither component a), i.e. a cell penetrating peptide, nor component c),i.e. a TLR peptide agonist.

Alternatively, however, the various antigens and/or antigenic epitopesmay also be positioned in any other way in the complex for use accordingto the present invention, for example with component a) and/or componentc) positioned in between two or more antigens and/or antigenic epitopes,i.e. with one or more antigens and/or antigenic epitopes positionedbetween component a) and component c) (or vice versa) and, optionally,one or more antigens and/or antigenic epitopes positioned at therespective other end of component a) and/or component c).

It is understood that a number of different antigens or antigenicepitopes relating to colorectal cancer may be advantageously comprisedby a single complex for use according to the present invention.Alternatively, a number of different antigens or antigenic epitopesrelating to colorectal cancer may be distributed to subsets of differentantigens or antigenic epitopes, in particular subsets complementing eachother in the context of colorectal cancer which are comprised bydifferent complexes according to the present invention, whereby suchdifferent complexes comprising different subsets may advantageously beadministered simultaneously, e.g. in a single vaccine, to a subject inneed thereof.

Preferably, the complex for use according to the present inventioncomprises at least one tumor epitope, which is an epitope of an antigenselected from the group consisting of EpCAM, HER-2, MUC-1, TOMM34, RNF43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5,COA-1, MAGE, SART and IL13Ralpha2. Those antigens are particularlyuseful in the context of colorectal cancer. It is also preferred thatthe complex for use according to the present invention comprises atleast one tumor antigen selected from the group consisting of EpCAM,HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2,p53, KRas, OGT, CASP5, COA-1, MAGE, SART and IL13Ralpha2, or a fragmentthereof, or a sequence variant of a tumor antigen or a sequence variantof a fragment thereof. As used herein, a “fragment” of an antigencomprises at least 10 consecutive amino acids of the antigen, preferablyat least 15 consecutive amino acids of the antigen, more preferably atleast 20 consecutive amino acids of the antigen, even more preferably atleast 25 consecutive amino acids of the antigen and most preferably atleast 30 consecutive amino acids of the antigen. A “sequence variant” isas defined above, namely a sequence variant has an (amino acid) sequencewhich is at least 70%, at least 75%, preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, particularlypreferably at least 95%, most preferably at least 99% identical to thereference sequence. A “functional” sequence variant means in the contextof an antigen/antigen fragment/epitope, that the function of theepitope(s), e.g. comprised by the antigen (fragment), is not impaired orabolished. Preferably, however, the amino acid sequence of theepitope(s), e.g. comprised by the cancer/tumor antigen (fragment) asdescribed herein, is not mutated and, thus, identical to the referenceepitope sequence.

As described above, suitable cancer/tumor epitopes of those antigens areknown from the literature or can be identified by using cancer/tumorepitope databases, e.g. from van der Bruggen P, Stroobant V, Vigneron N,Van den Eynde B. Peptide database: T cell-defined tumor antigens. CancerImmun 2013; URL: www.cancerimmunity.org/peptide/, wherein human tumorantigens recognized by CD4+ or CD8+ T cells are classified into fourmajor groups on the basis of their expression pattern, or from thedatabase “Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: cvc.dfci.harvard.edu/tadb/).

EpCAM

Ep-Cam is a glycoprotein mediating cellular adhesion. The amino acidsequence of EpCAM is shown in the following:

[SEQ ID NO: 47] MAPPQVLAFGLLLAAATATFAAAQEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAVVAGIVVLVISRKKRM AKYEKAEIKEMGEMHRELNA

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 47 ora fragment or a variant thereof as described herein.

Several epitopes of EpCAM are known to the skilled person. A preferredEpCAM epitope, which is preferably comprised by the complex for useaccording to the present invention, includes the following epitope (theepitope sequence shown in the following is a fragment of the above EpCAMsequence and is, thus, shown in the above EpCAM sequence underlined; thefollowing epitope sequence may refer to one epitope or more than one(overlapping) epitopes):

[SEQ ID NO: 48] GLKAGVIAV

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 48 ora fragment or a variant thereof as described herein.

HER-2/neu

Her-2 belongs to the EGFR (epidermal growth factor receptor) family.Many HLA-A epitopes are known to the skilled person. The amino acidsequence of HER2 is shown in the following:

[SEQ ID NO: 70] MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGA PPSTFKGTPTAENPEYLGLDVPV

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 70 ora fragment or a variant thereof as described herein.

As described above, suitable cancer/tumor epitopes of Her-2 are knownfrom the literature or can be identified by using cancer/tumor epitopedatabases, e.g. from van der Bruggen P, Stroobant V, Vigneron N, Van denEynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun2013; URL: www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: cvc.dfci.harvard.edu/tadb/).

Mucin-1 (MUC-1)

MUC-1 is a human epithelial mucin, acting on cell adhesion. The aminoacid sequence of MUC-1 is shown in the following:

[SEQ ID NO: 49] MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKV SAGNGGSSLSYTNPAVAATSANL

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 49 ora fragment or a variant thereof as described herein.

Several epitopes of MUC-1 are known to the skilled person. PreferredMUC-1 epitopes, which are preferably comprised by the complex for useaccording to the present invention, include the following epitopes (theepitope sequences shown in the following are fragments of the aboveMUC-1 sequence and are, thus, shown in the above MUC-1 sequenceunderlined; each of the following epitope sequences may refer to oneepitope or more than one (overlapping) epitopes):

[SEQ ID NO: 50] GSTAPPVHN [SEQ ID NO: 51] TAPPAHGVTS

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 50and/or an amino acid sequence according to SEQ ID NO: 51.

TOMM34

TOMM34 is involved in the import of precursor proteins intomitochondria. Many epitopes thereof are known to the skilled person.

RNF 43

RNF43 is a RING-type E3 ubiquitin ligase and is predicted to contain atransmembrane domain, a protease-associated domain, an ectodomain, and acytoplasmic RING domain. RNF43 is thought to negatively regulate Wntsignaling, and expression of RNF43 results in an increase inubiquitination of frizzled receptors, an alteration in their subcellulardistribution, resulting in reduced surface levels of these receptors.Many epitopes thereof are known to the skilled person.

KOC1

KOC1, also known as insulin-like growth factor 2 mRNA-binding protein3(IGF2BP3) is an mRNA binding protein. No expression data are howeveravailable.

Vascular Endothelial Growth Factor (VEGF)/Vascular Endothelial GrowthFactor Receptor (VEGFR)

Vascular endothelial growth factor (VEGF), originally known as vascularpermeability factor (VPF), is a signal protein produced by cells thatstimulates vasculogenesis and angiogenesis. It is part of the systemthat restores the oxygen supply to tissues when blood circulation isinadequate. VEGF's normal function is to create new blood vessels duringembryonic development, new blood vessels after injury, muscle followingexercise, and new vessels (collateral circulation) to bypass blockedvessels. There are three main subtypes of the receptors for VEGF(VEGFR), namely VEGFR1, VEGFR2 and VEGFR3.

Beta Subunit of Human Chorionic Gonadotropin (βhCG)

Human chorionic gonadotropin (hCG) is a hormone produced by the embryofollowing implantation. Some cancerous tumors produce this hormone;therefore, elevated levels measured when the patient is not pregnant canlead to a cancer diagnosis. hCG is heterodimeric with an a (alpha)subunit identical to that of luteinizing hormone (LH),follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH),and β (beta) subunit that is unique to hCG. The β-subunit of hCGgonadotropin (beta-hCG) contains 145 amino acids and is encoded by sixhighly homologous genes.

Survivin

Survivin, also called baculoviral inhibitor of apoptosisrepeat-containing 5 or BIRC5, is a member of the inhibitor of apoptosis(IAP) family. The survivin protein functions to inhibit caspaseactivation, thereby leading to negative regulation of apoptosis orprogrammed cell death. The amino acid sequence of survivin is shown inthe following:

[SEQ ID NO: 52] MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAKKVR RAIEQLAAMD

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 52 ora fragment or a variant thereof as described herein.

Several epitopes of survivinare known to the skilled person. A preferredsurvivin epitope, which is preferably comprised by the complex for useaccording to the present invention, includes the following epitope (theepitope sequence shown in the following is a fragment of the abovesurvivin sequence and is, thus, shown in the above survivin sequenceunderlined; the following epitope sequence may refer to one epitope ormore than one (overlapping) epitopes):

RISTFKNWPF

[SEQ ID NO: 53]

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 53.

Carcino-EMBRYONIC ANTIGEN (CEA)

CEA is an intracellular adhesion glycoprotein. CEA is normally producedin gastrointestinal tissue during fetal development, but the productionstops before birth. Therefore, CEA is usually present only at very lowlevels in the blood of healthy adults. The amino acid sequence of CEA isshown in the following:

[SEQ ID NO: 54] MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVAL

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 54 ora fragment or a variant thereof as described herein.

Several epitopes of CEA are known to the skilled person. Preferred CEAepitopes, which are preferably comprised by the complex for useaccording to the present invention, include the following epitopes (theepitope sequences shown in the following are fragments of the above CEAsequence and are, thus, shown in the above CEA sequence underlined; eachof the following epitope sequences may refer to one epitope or more thanone (overlapping) epitopes):

[SEQ ID NO: 55] YLSGANLNLS [SEQ ID NO: 56] SWRINGIPQQ

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 55and/or an amino acid sequence according to SEQ ID NO: 56.

Transforming Growth Factor Beta Receptor 2 (TGFβR2)

TGFβ receptors are single pass serine/threonine kinase receptors. Theyexist in several different isoforms. TGFβR2 is a transmembrane proteinthat has a protein kinase domain, forms a heterodimeric complex withanother receptor protein, and binds TGF-beta. This receptor/ligandcomplex phosphorylates proteins, which then enter the nucleus andregulate the transcription of a subset of genes related to cellproliferation.

P53

P53 is a tumor suppressor protein having a role in preventing genomemutation. P53 has many mechanisms of anticancer function and plays arole in apoptosis, genomic stability, and inhibition of angiogenesis. Inits anti-cancer role, p53 works through several mechanisms: it anactivate DNA repair proteins when DNA has sustained damage; it canarrest growth by holding the cell cycle at the G 1/S regulation point onDNA damage recognition; and it can initiate apoptosis.

Kirsten Ras (KRas)

GTPase KRas also known as V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog and KRAS, performs an essential function in normal tissuesignaling, and the mutation of a KRAS gene is an essential step in thedevelopment of many cancers. Like other members of the ras subfamily,the KRAS protein is a GTPase and is an early player in many signaltransduction pathways. KRAS is usually tethered to cell membranesbecause of the presence of an isoprene group on its C-terminus. Theamino acid sequence of KRas is shown in the following:

[SEQ ID NO: 57] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEK TPGCVKIKKCIIM

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 57 ora fragment or a variant thereof as described herein.

Several epitopes of Kirsten Ras are known to the skilled person. Apreferred Kirsten Ras epitope, which is preferably comprised by thecomplex for use according to the present invention, includes thefollowing epitope (the epitope sequence shown in the following is afragment of the above Kirsten Ras sequence and is, thus, shown in theabove Kirsten Ras sequence underlined; the following epitope sequencemay refer to one epitope or more than one (overlapping) epitopes):

[SEQ ID NO: 58] VVVGAGGVG

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 58.

O-Linked N-Acetylglucosamine (GlcNAc) Transferase (OGT)

OGT (O-Linked N-Acetylglucosamine (GlcNAc) Transferase, O-GlcNActransferase, OGTase, O-linked N-acetylglucosaminyl transferase, uridinediphospho-N-acetylglucosamine:polypeptidebeta-N-acetylglucosaminyltransferase, protein O-linkedbeta-N-acetylglucosamine transferase) is an enzyme with system nameUDP-N-acetyl-D-glucosamine:protein-O-beta-N-acetyl-D-glucosaminyltransferase) is an enzyme with system name“UDP-N-acetyl-D-glucosamine:protein-O-beta-N-acetyl-D-glucosaminyltransferase”. OGT catalyzes the addition of a single N-acetylglucosaminein O-glycosidic linkage to serine or threonine residues of intracellularproteins. OGT is a part of a host of biological functions within thehuman body. OGT is involved in the resistance of insulin in muscle cellsand adipocytes by inhibiting the Threonine 308 phosphorylation of AKT1,increasing the rate of IRS1 phosphorylation (at Serine 307 and Serine632/635), reducing insulin signaling, and glycosylating components ofinsulin signals. Additionally, OGT catalyzes intracellular glycosylationof serine and threonine residues with the addition ofN-acetylglucosamine. Studies show that OGT alleles are vital forembryogenesis, and that OGT is necessary for intracellular glycosylationand embryonic stem cell vitality. OGT also catalyzes theposttranslational modification that modifies transcription factors andRNA polymerase II, however the specific function of this modification ismostly unknown.

Caspase 5 (CASP5)

Caspase 5 is an enzyme that proteolytically cleaves other proteins at anaspartic acid residue, and belongs to a family of cysteine proteasescalled caspases. It is an inflammatory caspase, along with caspase 1,caspase 4 and the murine caspase 4 homolog caspase 11, and has a role inthe immune system.

Colorectal Tumor-Associated Antigen-1 (COA-1)

COA-1 was identified in 2003 by Maccalli et al. (Maccalli, C., et al.,Identification of a colorectal tumor-associated antigen (COA-1)recognized by CD4(+) T lymphocytes. Cancer Res, 2003. 63(20): p.6735-43) as strongly expressed by colorectal and melanoma cells (no dataavailable). Its mutation may interfere with the differential recognitionof tumor and normal cells.

Melanoma-Associated Antigen (MAGE)

The mammalian members of the MAGE (melanoma-associated antigen) genefamily were originally described as completely silent in normal adulttissues, with the exception of male germ cells and, for some of them,placenta. By contrast, these genes were expressed in various kinds oftumors. Therefore, the complex for use according to the presentinvention preferably comprises an antigen of the MAGE-family (a “MAGE”antigen) or an epitope thereof. Of the MAGE family, in particularMAGE-A3 and MAGE-D4 are preferred, and MAGE-A3 is particularlypreferred. The normal function of MAGE-A3 in healthy cells is unknown.MAGE-A3 is a tumor-specific protein, and has been identified on manytumors. The amino acid sequence of MAGE-A3 is shown in the following:

[SEQ ID NO: 59] MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKAFSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHIS YPPLHEW VLREGEE

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 59.

Several epitopes of MAGE-A3 are known to the skilled person. A preferredMAGE-A3 epitope, which is preferably comprised by the complex for useaccording to the present invention, includes the following epitope (theepitope sequence shown in the following is a fragment of the aboveMAGE-A3 sequence and is, thus, shown in the above MAGE-A3 sequenceunderlined; the following epitope sequence may refer to one epitope ormore than one (overlapping) epitopes):

[SEQ ID NO: 60] KVAELVHFL

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 60.

Squamous Cell Carcinoma Antigen Recognized by T-Cells (SART)

Within the SART family, SART-3 is most preferred. Thus, the complex foruse according to the present invention preferably comprises an antigenof the SART-family (a “SART” antigen) or an epitope thereof; the complexfor use according to the present invention more preferably comprisesSART-3 or an epitope thereof. Squamous cell carcinoma antigen recognizedby T-cells 3 possesses tumor epitopes capable of inducingHLA-A24-restricted and tumor-specific cytotoxic T lymphocytes in cancerpatients. SART-3 is thought to be involved in the regulation of mRNAsplicing.

IL13Ralpha2

IL13Ralpha2 binds interleukin 13 (IL-13) with very high affinity (andcan therefore sequester it) but does not allow IL-4 binding. It acts asa negative regulator of both IL-13 and IL-4, however the mechanism ofthis is still undetermined. The amino acid sequence of IL13Ralpha2 isshown in the following:

[SEQ ID NO: 61] MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQDFEIVDPGYLGYLYLQWQPPLSLDHFKECTVEYELKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEVQSSWAETTYWISPQGIPETKVQDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDHALQCVDYIKADGQNIGCRFPYLEASDYKDFYICVNGSSENKPIRSSYFTFQLQNIVKPLPPVYLTFTRESSCEIKLKWSIPLGPIPARCFDYEIEIREDDTTLVTATVENETYTLKTTNETRQLCFVVRSKVNIYCSDDGIWSEWSDKQCWEGEDLSKKTLLRFWLPFGFILILVIFVTG LLLRKPNTYPKMIPEFFCDT

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 61 ora fragment or a variant thereof as described herein.

Several epitopes of IL13Ralpha2are known to the skilled person. Apreferred IL13Ralpha2 epitope, which is preferably comprised by thecomplex for use according to the present invention, includes thefollowing epitope (the epitope sequence shown in the following is afragment of the above IL13Ralpha2 sequence and is, thus, shown in theabove IL13Ralpha2 sequence underlined; the following epitope sequencemay refer to one epitope or more than one (overlapping) epitopes):

[SEQ ID NO: 62] LPFGFIL

Accordingly, a preferred complex for use according to the presentinvention comprises an amino acid sequence according to SEQ ID NO: 62.

Preferably, the complex for use according to the present inventioncomprises at least one tumor epitope, which is an epitope of an antigenselected from the group consisting of EpCAM, MUC-1, survivin, CEA, KRas,MAGE-A3 and IL13Ralpha2, such as an epitope according to any of SEQ IDNOs 48, 50, 51, 53, 55, 56, 58, 60 and 62; more preferably the at leastone tumor epitope is an epitope of an antigen selected from the groupconsisting of EpCAM, MUC-1, survivin, CEA, KRas and MAGE-A3, such as anepitope according to any of SEQ ID NOs 48, 50, 51, 53, 55, 56, 58 and60; and even more preferably the at least one tumor epitope is anepitope of an antigen selected from the group consisting of EpCAM,MUC-1, survivin and CEA, such as an epitope according to any of SEQ IDNOs 48, 50, 51, 53, 55 and 56.

It is also preferred that the complex for use according to the presentinvention comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof;    -   one or more epitopes of KRas (such as the epitope according to        SEQ ID NO: 58) or functional sequence variants thereof; and/or    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

As described above, further epitopes of those antigens (in addition tothe exemplified epitopes) can easily be retrieved from cancer/tumorepitope databases, e.g. from van der Bruggen P, Stroobant V, Vigneron N,Van den Eynde B. Peptide database: T cell-defined tumor antigens. CancerImmun 2013; URL: www.cancerimmunity.org/peptide/, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: cvc.dfci.harvard.edu/tadb/).

A “sequence variant” is as defined above, namely a sequence variant hasan (amino acid) sequence which is at least 70%, at least 75%, preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, particularly preferably at least 95%, most preferably atleast 99% identical to the reference sequence. A “functional” sequencevariant means in the context of an epitope, that the function as anepitope is not impaired or abolished. Preferably, however, the aminoacid sequence of an epitope of a cancer/tumor antigen as describedherein is not mutated and, thus, identical to the reference epitopesequence.

It is also preferred that the complex for use according to the presentinvention comprises

-   -   a fragment of EpCAM comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of MUC-1 comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of survivin comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of CEA comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of KRas comprising one or more epitopes or a        functional sequence variant thereof; and/or    -   a fragment of MAGE-A3 comprising one or more epitopes or a        functional sequence variant thereof.

As used herein, a “fragment” of an antigen comprises at least 10consecutive amino acids of the antigen, preferably at least 15consecutive amino acids of the antigen, more preferably at least 20consecutive amino acids of the antigen, even more preferably at least 25consecutive amino acids of the antigen and most preferably at least 30consecutive amino acids of the antigen. Accordingly, a fragment of EpCAMcomprises at least 10 consecutive amino acids of EpCAM (SEQ ID NO: 47),preferably at least 15 consecutive amino acids of EpCAM (SEQ ID NO: 47),more preferably at least 20 consecutive amino acids of EpCAM (SEQ ID NO:47), even more preferably at least 25 consecutive amino acids of EpCAM(SEQ ID NO: 47) and most preferably at least 30 consecutive amino acidsof EpCAM (SEQ ID NO: 47); a fragment of MUC-1 comprises at least 10consecutive amino acids of MUC-1 (SEQ ID NO: 49), preferably at least 15consecutive amino acids of MUC-1 (SEQ ID NO: 49), more preferably atleast 20 consecutive amino acids of MUC-1 (SEQ ID NO: 49), even morepreferably at least 25 consecutive amino acids of MUC-1 (SEQ ID NO: 49)and most preferably at least 30 consecutive amino acids of MUC-1 (SEQ IDNO: 49); a fragment of survivin comprises at least 10 consecutive aminoacids of survivin (SEQ ID NO: 52), preferably at least 15 consecutiveamino acids of survivin (SEQ ID NO: 52), more preferably at least 20consecutive amino acids of survivin (SEQ ID NO: 52), even morepreferably at least 25 consecutive amino acids of survivin (SEQ ID NO:52) and most preferably at least 30 consecutive amino acids of survivin(SEQ ID NO: 52); a fragment of CEA comprises at least 10 consecutiveamino acids of CEA (SEQ ID NO: 54), preferably at least 15 consecutiveamino acids of CEA (SEQ ID NO: 54), more preferably at least 20consecutive amino acids of CEA (SEQ ID NO: 54), even more preferably atleast 25 consecutive amino acids of CEA (SEQ ID NO: 54) and mostpreferably at least 30 consecutive amino acids of CEA (SEQ ID NO: 54); afragment of KRas comprises at least 10 consecutive amino acids of KRas(SEQ ID NO: 57), preferably at least 15 consecutive amino acids of KRas(SEQ ID NO: 57), more preferably at least 20 consecutive amino acids ofKRas (SEQ ID NO: 57), even more preferably at least 25 consecutive aminoacids of KRas (SEQ ID NO: 57) and most preferably at least 30consecutive amino acids of KRas (SEQ ID NO: 57); and a fragment ofMAGE-A3 comprises at least 10 consecutive amino acids of MAGE-A3 (SEQ IDNO: 59), preferably at least 15 consecutive amino acids of MAGE-A3 (SEQID NO: 59), more preferably at least 20 consecutive amino acids ofMAGE-A3 (SEQ ID NO: 59), even more preferably at least 25 consecutiveamino acids of MAGE-A3 (SEQ ID NO: 59) and most preferably at least 30consecutive amino acids of MAGE-A3 (SEQ ID NO: 59).

A functional sequence variant of such a fragment has an (amino acid)sequence, which is at least 70%, at least 75%, preferably at least 80%,more preferably at least 85%, even more preferably at least 90%,particularly preferably at least 95%, most preferably at least 99%identical to the reference sequence, and the epitope function of atleast one, preferably all, epitope(s) comprised by the fragment ismaintained.

Preferably, such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof; and    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2, MUC-1,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT,CASP5, COA-1, SART or IL13Ralpha2.

It is also preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof; and    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT,CASP5, COA-1, SART or IL13Ralpha2.

It is also preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof; and    -   one or more epitopes of KRas (such as the epitope according to        SEQ ID NO: 58) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, OGT, CASP5,COA-1, MAGE, SART or IL13Ralpha2.

It is also preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof; and    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2, MUC-1,TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1,SART or IL13Ralpha2.

It is also preferred that such a complex comprises

-   -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof; and    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of EpCAM, HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, CEA, TGFβR2, p53, KRas, OGT, CASP5,COA-1, SART or IL13Ralpha2.

More preferably, such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof;        and/or    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1,MAGE, SART or IL13Ralpha2.

Particularly preferably, such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof; and    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1,MAGE, SART or IL13Ralpha2.

It is also particularly preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof; and    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT,CASP5, COA-1, MAGE, SART or IL13Ralpha2.

It is also particularly preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof; and    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2, MUC-1,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT,CASP5, COA-1, MAGE, SART or IL13Ralpha2.

It is also particularly preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2, MUC-1,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas,OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

It is also particularly preferred that such a complex comprises

-   -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof

Such a complex does preferably not comprise any epitope of EpCAM, HER-2,MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas,OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Component c)—TLR Peptide Agonist

In the complex for use according to the present invention, the TLRpeptide agonist allows an increased targeting of the vaccine towardsdendritic cells along with self-adjuvancity. Physical linkage of a TLRpeptide agonist to the CPP and the at least one antigen or antigenicepitope according to the present invention in the complex for useaccording to the present invention provides an enhanced immune responseby simultaneous stimulation of antigen presenting cells, in particulardendritic cells, that internalize, metabolize and display antigen(s).

As used in the context of the present invention, a “TLR peptide agonist”is an agonist of a Toll-like receptor (TLR), i.e. it binds to a TLR andactivates the TLR, in particular to produce a biological response.Moreover, the TLR peptide agonist is a peptide, a polypeptide or aprotein as defined above. Preferably, the TLR peptide agonist comprisesfrom 10 to 150 amino acids, more preferably from 15 to 130 amino acids,even more preferably from 20 to 120 amino acids, particularly preferablyfrom 25 to 110 amino acids, and most preferably from 30 to 100 aminoacids.

Toll like receptors (TLRs) are transmembrane proteins that arecharacterized by extracellular, transmembrane, and cytosolic domains.The extracellular domains containing leucine-rich repeats (LRRs) withhorseshoe-like shapes are involved in recognition of common molecularpatterns derived from diverse microbes. Toll like receptors includeTLRs1-10. Compounds capable of activating TLR receptors andmodifications and derivatives thereof are well documented in the art.TLR1 may be activated by bacterial lipoproteins and acetylated formsthereof, TLR2 may in addition be activated by Gram positive bacterialglycolipids, LPS, LP A, LTA, fimbriae, outer membrane proteins, heatshock proteins from bacteria or from the host, and Mycobacteriallipoarabinomannans. TLR3 may be activated by dsRNA, in particular ofviral origin, or by the chemical compound poly(LC). TLR4 may beactivated by Gram negative LPS, LTA, Heat shock proteins from the hostor from bacterial origin, viral coat or envelope proteins, taxol orderivatives thereof, hyaluronan containing oligosaccharides andfibronectins. TLR5 may be activated with bacterial flagellae orflagellin. TLR6 may be activated by mycobacterial lipoproteins and groupB streptococcus heat labile soluble factor (GBS-F) or staphylococcusmodulins. TLR7 may be activated by imidazoquinolines. TLR9 may beactivated by unmethylated CpG DNA or chromatin-IgG complexes.

Preferably, the TLR peptide agonist comprised by the complex for useaccording to the present invention is an agonist of TLR 1, 2, 4, 5, 6,and/or 10. TLRs are expressed either on the cell surface (TLR 1, 2, 4,5, 6, and 10) or on membranes of intracellular organelles, such asendosomes (TLR3, 4, 7, 8, and 9). The natural ligands for the endosomalreceptors turned out to be nucleic acid-based molecules (except forTLR4). The cell surface-expressed TLR 1, 2, 4, 5, 6, and 10 recognizemolecular patterns of extracellular microbes (Monie, T. P., Bryant, C.E., et al. 2009: Activating immunity: Lessons from the TLRs and NLRs.Trends Biochem. Sci. 34(11), 553-561). TLRs are expressed on severalcell types but virtually all TLRs are expressed on DCs allowing thesespecialized cells to sense all possible pathogens and danger signals.

However, TLR2, 4, and 5 are constitutively expressed at the surface ofDCs. Accordingly, the TLR peptide agonist comprised by the complex foruse according to the present invention is more preferably a peptideagonist of TLR2, TLR4 and/or TLR5. Even more preferably, the TLR peptideagonist is a TLR2 peptide agonist and/or a TLR4 peptide agonist.Particularly preferably, the TLR peptide agonist is a TLR4 peptideagonist. Most preferably, the TLR peptide agonist is one TLR peptideagonist, which is both, a TLR2 and a TLR4 agonist. TLR2 can detect awide variety of ligands derived from bacteria, viruses, parasites, andfungi. The ligand specificity is often determined by the interaction ofTLR2 with other TLRs, such as TLR1, 6, or 10, or non-TLR molecules, suchas dectin-1, CD14, or CD36. The formation of a heterodimer with TLR1enables TLR2 to identify triacyl lipoproteins or lipopeptides from(myco)bacterial origin, such as Pam3CSK4 and peptidoglycan (PGA; Gay, N.J., and Gangloff, M. (2007): Structure and function of Toll receptorsand their ligands. Annu. Rev. Biochem. 76, 141-165; Spohn, R.,Buwitt-Beckmann, U., et al. (2004): Synthetic lipopeptide adjuvants andToll-like receptor 2—Structure-activity relationships. Vaccine 22(19),2494-2499). Heterodimerization of TLR2 and 6 enables the detection ofdiacyl lipopeptides and zymosan. Lipopolysaccharide (LPS) and itsderivatives are ligands for TLR4 and flagellin for TLR5 (Bryant, C. E.,Spring, D. R., et al. (2010). The molecular basis of the host responseto lipopolysaccharide. Nat. Rev. Microbiol. 8(1), 8-14).

TLR2 interacts with a broad and structurally diverse range of ligands,including molecules expressed by microbes and fungi. Multiple TLR2agonists have been identified, including natural and syntheticlipopeptides (e.g. Mycoplasma fermentas macrophage-activatinglipopeptide (MALP-2)), peptidoglycans (PG such as those from S. aureus),lipopolysaccharides from various bacterial strains (LPS),polysaccharides (e.g. zymosan), glycosylphosphatidyl-inositol-anchoredstructures from gram positive bacteria (e.g. lipoteichoic acid (LTA) andlipo-arabinomannan from mycobacteria and lipomannas from M.tuberculosis). Certain viral determinants may also trigger via TLR2(Barbalat R, Lau L, Locksley R M, Barton G M. Toll-like receptor 2 oninflammatory monocytes induces type I interferon in response to viralbut not bacterial ligands. Nat Immunol. 2009: 10(11):1200-7). Bacteriallipopeptides are structural components of cell walls. They consist of anacylated s-glycerylcysteine moiety to which a peptide can be conjugatedvia the cysteine residue. Examples of TLR2 agonists, which are bacteriallipopeptides, include MALP-2 and it's synthetic analoguedi-palmitoyl-S-glyceryl cysteine (Pam₂Cys) or tri-palmitoyl-S-glycerylcysteine (Pam₃Cys).

A diversity of ligands interact with TLR4, including MonophosphorylLipid A from Salmonella minnesota R595 (MPLA), lipopolysaccharides(LPS), mannans (Candida albicans), glycoinositolphospholipids(Trypanosoma), viral envelope proteins (RSV and MMTV) and endogenousantigens including fibrinogen and heat-shock proteins. Such agonists ofTLR4 are for example described in Akira S, Uematsu S, Takeuchi O.Pathogen recognition and innate immunity. Cell. Feb 24; 2006:124(4):783-801 or in Kumar H, Kawai T, Akira S. Toll-like receptors andinnate immunity. Biochem Biophys Res Commun. Oct. 30; 2009 388(4):621-5.LPS, which is found in the outer membrane of gram negative bacteria, isthe most widely studied of the TLR4 ligands. Suitable LPS-derived TLR4agonist peptides are described for example in WO 2013/120073 (A1).

TLR5 is triggered by a region of the flagellin molecule expressed bynearly all motile bacteria. Thus, flagellin, or peptides or proteinsderived from flagellin and/or variants or fragments of flagellin arealso suitable as TLR peptide agonists comprised by the complex for useaccording to the present invention.

Examples of TLR peptide agonists thus include the TLR2 lipopeptideagonists MALP-2, Pam₂Cys and Pam₃Cys or modifications thereof, differentforms of the TLR4 agonist LPS, e.g. N. meningitidis wild-type L3-LPS andmutant penta-acylated LpxL1-LPS, and the TLR5 agonist flagellin.

However, it is preferred that the TLR peptide agonist comprised by thecomplex for use according to the present invention is neither alipopeptide nor a lipoprotein, neither a glycopeptide nor aglycoprotein, more preferably, the TLR peptide agonist comprised by thecomplex for use according to the present invention is a classicalpeptide, polypeptide or protein as defined herein.

A preferred TLR2 peptide agonist is annexin II or an immunomodulatoryfragment thereof, which is described in detail in WO 2012/048190 A1 andU.S. patent application Ser. No. 13/033,154,6, in particular a TLR2peptide agonist comprising an amino acid sequence according to SEQ IDNO: 4 or SEQ ID NO: 7 of WO 2012/048190 A1 or fragments or variantsthereof are preferred.

Thereby, a TLR2 peptide agonist comprising or consisting of an aminoacid sequence according to SEQ ID NO: 15 or a sequence variant thereofas described above is particularly preferred as component c), i.e. asthe at least one TLR peptide agonist, comprised by the complex for useaccording to the present invention.

(TLR2 peptide agonist Anaxa) SEQ ID NO: 15STVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Regarding TLR4, TLR peptides agonists are particularly preferred, whichcorrespond to motifs that bind to TLR4, in particular (i) peptidesmimicking the natural LPS ligand (RS10: Gln-Glu-Ile-Asn-Ser-Ser-Tyr andRS09: Ala-Pro-Pro-His-Ala-Leu-Ser) and (ii) Fibronectin derivedpeptides. The cellular glycoprotein Fibronectin (FN) has multipleisoforms generated from a single gene by alternative splicing of threeexons. One of these isoforms is the extra domain A (EDA), whichinteracts with TLR4.

Further suitable TLR peptide agonists comprise a fibronectin EDA domainor a fragment or variant thereof. Such suitable fibronectin EDA domainsor a fragments or variants thereof are disclosed in EP 1 913 954 B1, EP2 476 440 A1, US 2009/0220532 A1, and WO 2011/101332 A1. Thereby, a TLR4peptide agonist comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 45 or a sequence variant thereof as describedabove is particularly preferred as component c), i.e. as the at leastone TLR peptide agonist, comprised by the complex for use according tothe present invention.

(TLR4 peptide agonist EDA) SEQ ID NO: 45NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIG IQST

In addition, high-mobility group box 1 protein (HMGB1) and peptidefragments thereof are assumed to be TLR4 agonists. Such HMGB1-derivedpeptides are for example disclosed in US 2011/0236406 A1.

The complex for use according to the present invention comprises atleast one TLR peptide agonist, preferably the complex for use accordingto the present invention comprises more than one TLR peptide agonist, inparticular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TLR peptide agonists, morepreferably the complex for use according to the present inventioncomprises (at least) two or three TLR peptide agonists, even morepreferably the complex for use according to the present inventioncomprises (at least) four or five TLR peptide agonists. If more than oneTLR peptide agonist is comprised by the complex for use according to thepresent invention it is understood that said TLR peptide agonist is inparticular also covalently linked in the complex for use according tothe present invention, e.g. to another TLR peptide agonist and/or to acomponent a), i.e. a cell penetrating peptide, and/or to a component b),i.e. an antigen or antigenic epitope.

In a particularly preferred embodiment, the complex for use according tothe present invention comprises one single TLR peptide agonist. Inparticularly, in this particularly preferred embodiment, the complex foruse according to the present invention comprises one single TLR peptideagonist and no further component having TLR agonist properties exceptthe one single TLR peptide agonist as described.

The various TLR peptide agonists comprised by the complex for useaccording to the present invention may be the same or different.Preferably, the various TLR peptide agonists comprised by the complexfor use according to the present invention are different from eachother.

Moreover, it is preferred that the more than one antigen or antigenicepitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 antigens or antigenicepitopes, or more TLR peptide agonists, in particular 2, 3, 4, 5, 6, 7,8, 9, 10 TLR agonists, are positioned consecutively in the complex foruse according to the present invention. This means in particular thatall TLR peptide agonists comprised by the complex are positioned in astretch, which is neither interrupted by component a), i.e. a cellpenetrating peptide, nor by component b), i.e. at least one antigen orantigenic epitope. Rather, component a) and component b) are positionedin the complex for example before or after such a stretch of all TLRpeptide agonists. However, the TLR peptide agonists positionedconsecutively in such a way may be linked to each other for example by aspacer or linker as described below, which is neither component a), i.e.a cell penetrating peptide, nor component b), i.e. at least one antigenor antigenic epitope.

Alternatively, however, the various TLR peptide agonists may also bepositioned in any other way in the complex for use according to thepresent invention, for example with component a) and/or component b)positioned in between two or more TLR peptide agonists, i.e. with one ormore TLR peptide agonist positioned between component a) and componentb) (or vice versa) and, optionally, one or more TLR peptide agonistspositioned at the respective other end of component a) and/or componentb).

It is understood that a number of different TLR peptide agonistsactivating the same or different TLR receptors may be advantageouslycomprised by a single complex for use according to the presentinvention. Alternatively, a number of different TLR peptide agonistsactivating the same or different TLR receptors may be distributed tosubsets of different TLR peptide agonists activating the same ordifferent TLR receptors, which are comprised by different complexesaccording to the present invention, whereby such different complexescomprising different subsets may advantageously be administeredsimultaneously, e.g. in a single vaccine, to a subject in need thereof.

Linkage of Components a), b), and c) in the Complex for Use According tothe Present Invention

In the complex for use according to the present invention, componentsa), b) and c) are covalently linked, i.e. the linkage between two out ofthe three components a), b), and c) of the complex for use according tothe present invention is a covalent linkage. Preferably, two out of thethree components a), b), and c) of the complex for use according to thepresent invention are covalently linked to each other (i.e. the “first”and the “second” component), and the third component out of the threecomponents a), b), and c) is covalently linked either to the firstcomponent out of the three components a), b), and c) or to the secondcomponent out of the three components a), b), and c). Thereby,preferably a linear molecule is formed. However, it is also conceivablethat each of the three components a), b), and c) is covalently linked toboth of the other components out of the three components a), b), and c).

A “covalent linkage” (also covalent bond), as used in the context of thepresent invention, refers to a chemical bond that involves the sharingof electron pairs between atoms. A “covalent linkage” (also covalentbond) in particular involves a stable balance of attractive andrepulsive forces between atoms when they share electrons. For manymolecules, the sharing of electrons allows each atom to attain theequivalent of a full outer shell, corresponding to a stable electronicconfiguration. Covalent bonding includes many kinds of interactions,including for example σ-bonding, π-bonding, metal-to-metal bonding,agostic interactions, and three-center two-electron bonds. Accordingly,the complex for use according to the present invention may also bereferred to as “compound”, in particular it may be referred to as“molecule”.

Preferably, in the complex for use according to the present invention,components a), b), and c) are covalently linked by chemical coupling inany suitable manner known in the art, such as cross-linking methods.However, attention is drawn to the fact that many known chemicalcross-linking methods are non-specific, i.e., they do not direct thepoint of coupling to any particular site on the components a), b), andc). Thus, the use of non-specific cross-linking agents may attackfunctional sites or sterically block active sites, rendering the fusedcomponents of the complex for use according to the present inventionbiologically inactive. It is referred to the knowledge of the skilledartisan to block potentially reactive groups by using appropriateprotecting groups. Alternatively, the use of the powerful and versatileoxime and hydrazone ligation techniques, which are chemo-selectiveentities that can be applied for the cross-linking of components a), b),and c) may be employed. This linking technology is described e.g. byRose et al. (1994), JACS 116, 30.

Coupling specificity can be increased by direct chemical coupling to afunctional group found only once or a few times in components a), b),and/or c), which functional group is to be cross-linked to the anotherof the components a), b), and c). As an example, the cystein thiol groupmay be used, if just one cystein residue is present in a certaincomponent a), b), or c) of complex for use according to the presentinvention. Also, for example, if a certain component a), b), or c)contains no lysine residues, a cross-linking reagent specific forprimary amines will be selective for the amino terminus of therespective component. Alternatively, cross-linking may also be carriedout via the side chain of a glutamic acid residue placed at theN-terminus of the peptide such that a amide bond can be generatedthrough its side-chain. Therefore, it may be advantageous to link aglutamic acid residue to the N-terminus of a certain component a), b),or c). However, if a cysteine residue is to be introduced into a certaincomponent a), b), or c), introduction at or near its N- or C-terminus ispreferred. Conventional methods are available for such amino acidsequence alterations based on modifications of certain component a), b),or c) by either adding one or more additional amino acids, e.g. interalia an cystein residue, to the translocation sequence or bysubstituting at least one residue of the translocation sequence(s) beingcomprised in the respective component. In case a cystein side chain isused for coupling purposes, a certain component a), b), or c) haspreferably one cystein residue. Any second cystein residue shouldpreferably be avoided and can, optionally, be replaced when they occurin the respective component comprised by the complex for use accordingto the present invention. When a cysteine residue is replaced in theoriginal sequence of a certain component a), b), or c), it is typicallydesirable to minimize resulting changes in the peptide folding of therespective component. Changes in folding are minimized when thereplacement is chemically and sterically similar to cysteine. Therefore,serine is preferred as a replacement for cystein.

Coupling of two out of the three components a), b), and c) can beaccomplished via a coupling or conjugating agent including standardpeptide synthesis coupling reagents such as HOBt, HBTU, DICI, TBTU.There are several intermolecular cross-linking agents which can beutilized, see for example, Means and Feeney, Chemical Modification ofProteins, Holden-Day, 1974, pp. 39-43. Among these reagents are, forexample, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) orN,N′-(1,3-phenylene)bismaleimide; N,N′-ethylene-bis-(iodoacetamide) orother such reagent having 6 to 11 carbon methylene bridges; and1,5-difluoro-2,4-dinitrobenzene. Other cross-linking agents useful forthis purpose include: p,p′-difluoro-m,m′-dinitrodiphenylsulfone;dimethyl adipimidate; phenol-1,4-disulfonylchloride;hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate; glutaraldehyde and disdiazobenzidine.Cross-linking agents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking agent is bismaleimidohexane (BMH). BMHcontains two maleimide functional groups, which react specifically withsulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). Thetwo maleimide groups are connected by a hydrocarbon chain. Therefore,BMH is useful for irreversible cross-linking of proteins (orpolypeptides) that contain cysteine residues. Cross-linking agents mayalso be heterobifunctional. Heterobifunctional cross-linking agents havetwo different functional groups, for example an amine-reactive group anda thiol-reactive group, that will cross-link two proteins having freeamines and thiols, respectively. Examples of heterobifunctionalcross-linking agents areSuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimide4-(p-maleimidophenyl)butyrate (SMPB), an extended chain analog of MBS.The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue. Because cross-linking agents often have lowsolubility in water, a hydrophilic moiety, such as a sulfonate group,may be added to the cross-linking agent to improve its water solubility.Sulfo-MBS and sulfo-SMCC are examples of cross-linking agents modifiedfor water solubility. Many cross-linking agents yield a conjugate thatis essentially non-cleavable under cellular conditions. Therefore, somecross-linking agents contain a covalent bond, such as a disulfide, thatis cleavable under cellular conditions. For example, Traut's reagent,dithiobis (succinimidylpropionate) (DSP), and N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) are well-known cleavablecross-linkers. The use of a cleavable cross-linking agent permits thecell penetrating peptide, the at least one antigen or antigenic epitopeand the at least one TLR peptide agonist comprised by the complex foruse according to the present invention to separate from each other afterdelivery into the target cell. For this purpose, direct disulfidelinkage may also be useful. Chemical cross-linking may also include theuse of spacer arms. Spacer arms provide intramolecular flexibility oradjust intramolecular distances between conjugated moieties and therebymay help preserve biological activity. A spacer arm may be in the formof a protein (or polypeptide) moiety that includes spacer amino acids,e.g. proline. Alternatively, a spacer arm may be part of thecross-linking agent, such as in “long-chain SPDP” (Pierce Chem. Co.,Rockford, Ill., cat. No. 21651 H). Numerous cross-linking agents,including the ones discussed above, are commercially available. Detailedinstructions for their use are readily available from the commercialsuppliers. More detailed information on protein cross-linking andconjugate preparation, which is useful in the context of linkage ofcomponents a), b), and c) comprised by the complex for use according tothe present invention can be retrieved from: Wong, Chemistry of ProteinConjugation and Cross-Linking, CRC Press (1991).

Cross-linking agents for peptide or protein crosslinking include forexample (i) amine-to-amine crosslinkers, e.g. homobifunctionalamine-specific protein crosslinking reagents based on NHS-ester andimidoester reactive groups for selective conjugation of primary amines;available in short, long, cleavable, irreversible, membrane permeable,and cell surface varieties; (ii) sulfhydryl-to-carbohydratecrosslinkers, e.g. crosslinking reagents based on maleimide andhydrazide reactive groups for conjugation and formation of covalentcrosslinks; (iii) sulfhydryl-to-sulfhydryl crosslinkers, e.g.homobifunctional sulfhydryl-specific crosslinking reagents based onmaleimide or pyridyldithiol reactive groups for selective covalentconjugation of protein and peptide thiols (reduced cysteines) to formstable thioether bonds; (iv) photoreactive crosslinkers, e.g. arylazide, diazirine, and other photo-reactive (light-activated) chemicalheterobifunctional crosslinking reagents to conjugate proteins, nucleicacids and other molecular structures involved in receptor-ligandinteraction complexes via two-step activation; (v) amine-to-sulfhydrylcrosslinkers, e.g. heterobifunctional protein crosslinking reagents forconjugation between primary amine (lysine) and sulfhydryl (cysteine)groups of proteins and other molecules; available with different lengthsand types of spacer arms; and (vi) amine-to-amine crosslinkers, e.g.carboxyl-to-amine crosslinkers, e.g. Carbodiimide crosslinking reagents,DCC and EDC (EDAC), for conjugating carboxyl groups (glutamate,aspartate, C-termini) to primary amines (lysine, N-termini) and alsoN-hydroxysuccinimide (NHS) for stable activation of carboxylates foramine-conjugation.

Examples of crosslinkers in general, which can be used in the complexfor use according to the present invention, includeN-(α-Maleimidoacetoxy)-succinimide ester,N-5-Azido-2-nitrobenzyloxy-succinimide, 1,4-Bis-Maleimidobutane,1,4-Bis-Maleimmidyl-2,3-dihydroxy-butane, Bis-Maleimidohexane,Bis-Maleimidoethane, N-(β-Maleimidopropionic acid)hydrazide*TFA,N-(β-Maleimidopropyloxy)succinimide ester,1,8-Bis-Maleimidodiethylene-glycol, 1,11-Bis-Maleimidotriethyleneglycol,Bis (sulfosuccinimidyl)suberate, Bis (sulfosuccinimidyl)glutarate-d0,Bis (sulfosuccinimidyl)2,2,4,4-glutarate-d4, Bis(sulfosuccinimidyl)suberate-d0, Bis(sulfosuccinimidyl)2,2,7,7-suberate-d4, Bis (NHS)PEG5, Bis (NHS)PEG9,Bis (2-[succinimidoxycarbonyloxy]ethyl)sulfone,N,N-Dicyclohexylcarbodiimide, 1-5-Difluoro-2,4-dinitrobenzene, Dimethyladipimidate*2HCI, Dimethyl pimelimidate*2HCI, Dimethylsuberimidate*2HCl, Disuccinimidyl glutarate,Dithiobis(succimidylpropionate) (Lomant's Reagent), Disuccinimidylsuberate, Disuccinimidyl tartarate, Dimethyl3,3′-dithiobispropionimidate*2HCI, Dithiobis-maleimidoethane,3,3′-Dithiobis (sulfosuccinimidylpropionate),1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, Ethyleneglycol bis (succinimidylsuccinate), N-ε-Maleimidocaproic acid,N-(ε-Maleimidocaproic acid)hydrazide,N-(ε-Maleimidocaproyloxy)succinimide ester,N-(γ-Maleimidobutyryloxy)succinimide ester, N-(κ-Maleimidoundecanoicacid)hydrazide, NHS-LC-Diazirine, Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate, Succinimidyl6-(3′-[2-pyridyldithio] propionamido)hexanoate, L-Photo-Leucine,L-Photo-Methionine, m-Maleimidobenzoyl-N-hydroxysuccinimide ester,4-(4-N-Maleimidophenyl)-butyric acid hydrazide*HCI,2-[N2-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-L-lysinyl]ethylmethanethiosulfate,2-{N2-[N6-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-L-lysinyl]}ethylmethanethiosulfate,N-Hydroxysuccinimide, N-hydroxysuccinimide ester ethane azide,N-hydroxysuccinimide ester tetraoxapentadecane azide,N-hydroxysuccinimide ester dodecaoxanonatriacontane azide,NHS-Phosphine, 3-(2-Pyridyldithio)propionylhydrazide,2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide,2-pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide,N-(ρ-Maleimidophenyl)isocyanate, Succinimdyl3-(bromoacetamido)propionate, NHS-Diazirine, NHS-SS-Diazirine,N-succinimidyl iodoacetate, N-Succinimidyl(4-iodoacetyl)aminobenzoate,Succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate,NHS-PEG2-Maliemide, NHS-PEG4-Maliemide, NHS-PEG6-Maleimide,NHS-PEG8-Maliemide, NHS-PEG12-Maliemide, NHS-PEG24-Maleimide,Succinimidyl 4-(ρ-maleimido-phenyl)butyrate,Succinimidyl-6-(β-maleimidopropionamido)hexanoate,4-Succinimidyloxycarbonyl-methyl-α-(2-pyridyldithio)toluene,Succinimidyl-(4-psoralen-8-yloxy)butyrate, N-Succinimidyl3-(2-pyridyldithio)propionate, Ethylene glycol bis (sulfo-succinimidylsuccinate), N-(ε-Maleimidocaproyloxy)sulfosuccinimide ester,N-(γ-Maleimidobutryloxy)sulfosuccinimide ester,N-(κ-Maleimidoundecanoyloxy)sulfosuccinimide ester,Sulfo-NHS-LC-Diazirine, Sulfosuccinimidyl6-(3′-[2-pyridyldithio]propionamido)hexanoate,m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester,N-Hydroxysuccinimide, Sulfo-NHS-Phosphine, Sulfosuccinimidyl6-(4′-azido-2′-nitrophenylamino)hexanoate,Sulfo-NHS-(2-6-[Biotinamido]-2-(ρ-azidobezamido), Sulfo-NHS-Diazirine,Sulfo-NHS-SS-Diazirine, Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate,Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,Sulfosuccinimidyl 4-(ρ-maleimidophenyl)butyrate,Tris-(2-Maleimidoethyl)amine (Trifunctional), and Tris-(succimimidylaminotricetate) (Trifunctional).

The linkage between two out of the three components a), b), and c) ofthe complex for use according to the present invention may be directlyor indirectly, i.e. two components directly adjoin or they may be linkedby an additional component of the complex, e.g. a spacer or a linker.

A direct linkage may be realized preferably by an amide bridge, if thecomponents to be linked have reactive amino or carboxy groups. Morespecifically, if the components to be linked are peptides, polypeptidesor proteins, a peptide bond is preferred. Such a peptide bond may beformed using a chemical synthesis involving both components (anN-terminal end of one component and the C-terminal end of the othercomponent) to be linked, or may be formed directly via a proteinsynthesis of the entire peptide sequence of both components, whereinboth (protein or peptide) components are preferably synthesized in onestep. Such protein synthesis methods include e.g., without being limitedthereto, liquid phase peptide synthesis methods or solid peptidesynthesis methods, e.g. solid peptide synthesis methods according toMerrifield, t-Boc solid-phase peptide synthesis, Fmoc solid-phasepeptide synthesis, BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate) based solid-phase peptide synthesis, etc. . . .Alternatively, ester or ether linkages are preferred.

Moreover, in particular if the components to be linked are peptides,polypeptides or proteins, a linkage may occur via the side chains, e.g.by a disulfide bridge. Further components of other chemical nature, e.g.the at least one antigen or antigenic epitope if it is not of peptidicnature, may be likewise attached to the components of peptidic nature,e.g. the cell penetrating peptide, the at least one TLR peptide agonist,and the at least one antigen or antigenic epitope if it is of peptidicnature. The linkage via a side chain will preferably be based on sidechain amino, thiol or hydroxyl groups, e.g. via an amide or ester orether linkage. A linkage of a peptidic main chain with a peptidic sidechain of another component may also be via an isopeptide bond. Anisopeptide bond is an amide bond that is not present on the main chainof a protein. The bond forms between the carboxyl terminus of onepeptide or protein and the amino group of a lysine residue on another(target) peptide or protein.

The complex for use according to the present invention may optionallycomprise a spacer or linker, which are non-immunologic moieties, whichare preferably cleavable, and which link component a) and b) and/orcomponent a) and c), and/or component b) and c), and/or link consecutiveantigens or antigenic epitopes, and/or link consecutive TLR peptideagonists, and/or link consecutive cell penetrating peptides, and/orwhich can be placed at the C-terminal part of components b) and/or c). Alinker or spacer may preferably provide further functionalities inaddition to linking of the components, and preferably being cleavable,more preferably naturally cleavable inside the target cell, e.g. byenzymatic cleavage. However, such further functionalities do inparticular not include any immunological functionalities. Examples offurther functionalities, inparticular regarding linkers in fusionproteins, can be found in Chen X. et al., 2013: Fusion Protein Linkers:Property, Design and Functionality. Adv Drug Deliv Rev. 65(10):1357-1369, wherein for example also invivo cleavable linkers aredisclosed. Moreover, Chen X. et al., 2013: Fusion Protein Linkers:Property, Design and Functionality. Adv Drug Deliv Rev. 65(10):1357-1369 also discloses various linkers, e.g. flexible linkers andrigid linkers, and linker designing tools and databases, which can beuseful in the complex for use according to the present invention or todesign a linker to be used in the complex for use according to thepresent invention.

Said spacer may be peptidic or non-peptidic, preferably the spacer ispeptidic. Preferably, a peptidic spacer consists of about 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 amino acids, more preferably of about 1, 2, 3, 4, or 5amino acids. The amino acid sequence of the peptidic spacer may beidentical to that of the N-terminal or C-terminal flanking region of anyof the components a), b), or c). Alternatively a peptidic spacer canconsist of non-natural amino acid sequences such as an amino acidsequence resulting from conservative amino acid substitutions of saidnatural flanking regions or sequences of known cleavage sites forproteases such as an enterokinase target site (amino acid sequence:DDDK, SEQ ID NO: 16), factor Xa target site (amino acid sequence: IEDGR,SEQ ID NO: 17), thrombin target site (amino acid sequence: LVPRGS, SEQID NO: 18), protease TEV target site (amino acid sequence: ENLYFQG, SEQID NO: 19), PreScission protease target site (amino acid sequenceLEVLFQGP, SEQ ID NO: 20), polycationic amino acids, e.g. poly K, furintarget site (amino acid sequence RX(R/K)R, SEQ ID NO: 21). In aparticular embodiment, the peptidic spacer does not contain any Cys (C)residues. In a preferred embodiment the linker sequence contains atleast 20%, more preferably at least 40% and even more preferably atleast 50% Gly or β-alanine residues, e.g. GlyGlyGlyGlyGly (SEQ ID NO:22), GlyGlyGlyGly (SEQ ID NO: 23), GlyGlyGly, CysGlyGly or GlyGlyCys,etc. Appropriate linker sequences can be easily selected and prepared bya person skilled in the art. They may be composed of D and/or L aminoacids. Further examples of a peptidic spacer include the amino acidsequences EQLE (SEQ ID NO: 24) or TEWT (SEQ ID NO: 25) or anyconservative substitutions thereof.

A non-peptidic spacer can include or may be an ester, a thioester, and adi-sulfide.

In particular, the complex for use according to the invention maycomprise a spacer or linker, in particular a peptidic spacer, placedbetween component a) and b) and/or between component a) and c), and/orbetween component b) and c),. This peptidic spacer can be chosen by oneskilled in the art so that it may be cut by the cell machinery once thecomplex comprising the cell penetrating peptide and the cargo moleculehas been internalized.

When the complex comprises several antigens or antigenic epitopes orwhen the complex comprises several TLR peptide agonists, it will beclear for one skilled in the art that each of the antigens or antigenicepitopes and/or each of the TLR peptide agonists comprised in thecomplex of the invention can be either directly linked to each other orlinked via spacers or linkers such as, e.g., a peptidic spacerconsisting of a few amino acids. Alternatively, when the complex for useaccording to the present invention comprises several antigens orantigenic epitopes or when the complex comprises several TLR peptideagonists, it is also possible that some antigens or antigenic epitopesand/or some TLR peptide agonists comprised by the complex of theinvention are directly linked to each other and some other antigens orantigenic epitopes and/or some other TLR peptide agonists are linked viaspacers or linkers such as a peptidic spacer consisting of a few aminoacids.

For example, two successive antigens or antigenic epitopes or twosuccessive TLR peptide agonists comprised in the complex of theinvention are linked to each other by spacers consisting of the naturalflanking regions of said antigens or antigenic epitopes or of said TLRpeptide agonists, respectively. For example, the spacer used to link afirst antigen/antigenic epitope or a first TLR peptide agonist to asecond antigen/antigenic epitope or to a second TLR peptide agonist,respectively, may consists of up to about 8 amino acids corresponding toup to about 4 amino acids of the N-terminal or C-terminal flankingregion of the first antigen/antigenic epitope or the first TLR peptideagonist, followed by up to about 4 amino acids of the N-terminal orC-terminal flanking region of the second antigen/antigenic epitope orthe second TLR peptide agonist. In an illustration of the presentinvention, the spacer used to link a first antigen/antigenic epitope ora first TLR peptide agonist (“antigen/epitope/TLR peptide agonist 1”) toa second epitope (“antigen/epitope/TLR peptide agonist 2”) consists ofabout 8 amino acids corresponding to any possible combination rangingfrom: 0 flanking amino acid of antigen/epitope/TLR peptide agonist 1 and8 flanking amino acids of antigen/epitope/TLR peptide agonist 2, to 8flanking amino acids of antigen/epitope/TLR peptide agonist 1 and 0flanking amino acid of antigen/epitope/TLR peptide agonist 2, i.e.including 1 flanking amino acid of antigen/epitope/TLR peptide agonistand 7 flanking amino acids of antigen/epitope/TLR peptide agonist 2, 2flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 6flanking amino acids of antigen/epitope/TLR peptide agonist 2, 3flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 5flanking amino acids of antigen/epitope/TLR peptide agonist 2, 4flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 4flanking amino acids of antigen/epitope/TLR peptide agonist 2, 5flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 3flanking amino acids of antigen/epitope/TLR peptide agonist 2, 6flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 2flanking amino acids of antigen/epitope/TLR peptide agonist 2, 7flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 1flanking amino acid of antigen/epitope/TLR peptide agonist 2, 8 flankingamino acid of antigen/epitope/TLR peptide agonist 1 and 0 flanking aminoacids of antigen/epitope/TLR peptide agonist 2. It will be understoodthat the total of 8 amino acids constituting a spacer linking twoconsecutive antigen/epitope/TLR peptide agonist is not an absolute valueand the spacer could also be composed of a total of, for instance, 3amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids,9 amino acids or 10 amino acids. Similarly, equivalent combinations asmentioned above are also an illustration of the invention in thesituation where a spacer has less or more than 8 amino acids.

In another particular illustration of the present invention, the spacerused to link a first antigen/antigenic epitope or a first TLR peptideagonist (“antigen/epitope/TLR peptide agonist 1”) to a secondantigen/antigenic epitope or to a second TLR peptide agonist,respectively, (“antigen/epitope/TLR peptide agonist 2”) consists of e.g.1, 2, 3, 4, or 5 amino acids. More particularly, said spacer's aminoacid sequence can correspond to the 4 amino acids of the N-terminal orC-terminal flanking region of antigen/epitope/TLR peptide agonist 1 orantigen/epitope/TLR peptide agonist 2. A spacer as described above mayalso be placed at the C-terminal part of the last antigen/epitope/TLRpeptide agonist comprised in the complex for use according to thepresent invention.

The technics for linking two of the three components a), b), and c) arewell documented in the literature and can depend on the nature of the atleast one antigen or antigenic epitope. For instance, linkages betweentwo of the three components a), b), and c) can be achieved via cleavabledisulphide linkages through total stepwise solid-phase synthesis orsolution-phase or solid-phase fragment coupling, stable amide,thiazolidine, oxime and hydrazine linkage, disulphide linkage, stablethiomaleimide linkage, peptide bond (including peptide bonds betweenamino acids of a fusion protein), or electrostatic or hydrophobicinteractions.

Preferably, the at least one antigen or antigenic epitope comprised bythe complex for use according to the present invention as well as anyoptional spacer or linker comprised by the complex for use according tothe present invention are of peptidic nature. More preferably, allcomponents of the complex for use according to the present invention,e.g. the cell penetrating peptide, the at least one antigen or antigenicepitope, which is a peptide, polypeptide or protein, the at least oneTLR peptide agonist and any optional peptidic linker or spacer arelinked in the complex for use according to the present invention by apeptide bond. Most preferably, the complex for use according to thepresent invention is thus a peptide, polypeptide or protein, such as afusion protein, e.g. a recombinant fusion protein.

In this context, a complex comprising or consisting of an amino acidsequence according to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46 or SEQ ID NO: 69 or acomplex comprising or consisting of an amino acid sequence sharing atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 98% sequence identitywith any of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO: 46 or SEQ ID NO: 69 is preferred; acomplex comprising or consisting of an amino acid sequence according toSEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 or a complexcomprising or consisting of an amino acid sequence sharing at least 50%,at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 98% sequence identity with any ofSEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is more preferred; acomplex comprising or consisting of an amino acid sequence according toSEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41 or SEQ ID NO: 69 or a complex comprising or consisting of an aminoacid sequence sharing at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least98% sequence identity with SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is even more preferred;and a complex comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41or SEQ ID NO: 69 or a complex comprising or consisting of an amino acidsequence sharing at least 50%, at least 60%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95% or at least 98%sequence identity with SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41 or SEQ ID NO: 69 is particularly preferred.

SEQ ID NO: 26: MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGEDDTAELQGLR PGSEYTVSVVALHDDMESQP LIGIQSTKRY KNRVASRKSR AKFKQLLQHYREVAAAKSSE NDRLRLLLKE SLKISQAVHA AHAEINEAGREVVGVGALKV PRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLE SIINFEKLTE WTGSSEQ ID NO: 27: MHHHHHHSTV HEILCKLSLE GDHSTPPSAY GSVKPYTNFDAEKRYKNRVA SRKSRAKFKQ LLQHYREVAA AKSSENDRLRLLLKESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQDWLGVPRFAKF ASFEAQGALA NIAVDKANLD VEQLESIINF EKLTEWTGS SEQ ID NO: 28:MHHHHHHKRYKNRVA SRKSRAKFKQ LLQHYREVAAAKSSENDRLR LLLKESLKIS QAVHAAHAEI NEAGREVVGVGALKVPRNQD WLGVPRFAKF ASFEAQGALA NIAVDKANLDVEQLESIINF EKLTEWTGSS TVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAESEQ ID NO: 33: MHHHHHHKRY KNRVASRKSR AKFKQLLQHY REVAAAKESLKISQAVHAAH AEINEAGREV VGVGALKVPR NQDWLGVPRFAKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWTGSSTVHEILC KLSLEGDHST PPSAYGSVKP YTNFDAE SEQ ID NO: 34:MHHHHHHREV AAAKSSENDR LRLLLKESLK ISQAVHAAHAEINEAGREVV GVGALKVPRN QDWLGVPRFA KFASFEAQGALANIAVDKAN LDVEQLESII NFEKLTEWTG SSTVHEILCK LSLEGDHSTP PSAYGSVKPY TNFDAESEQ ID NO: 37: MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGE DDTAELQGLR PGSEYTVSVVALHDDMESQP LIGIQSTKRY KNRVASRKSR AKFKQLLQHYREVAAAKESL KISQAVHAAH AEINEAGREV VGVGALKVPRNQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWT GS SEQ ID NO: 38:MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGE DDTAELQGLR PGSEYTVSVVALHDDMESQP LIGIQSTREV AAAKSSENDR LRLLLKESLKISQAVHAAHA EINEAGREVV GVGALKVPRN QDWLGVPRFAKFASFEAQGA LANIAVDKAN LDVEQLESII NFEKLTEWTG S SEQ ID NO: 39:KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKVTYHSPSYAYHQFERRAILNRLVQFIKDRISVVQALVLTSTVHEILCKLSLEGDHSTPPSAYGSVKPYTN FDAE SEQ ID NO: 40:KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNYRIATFKNWPFLEDCAMEELTVSEFLKLDRQRSTVHEILCKLS LEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 41: KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKHLELASMTNMELMSSIVSTVHEILCKLSLEGDHSTPPSAYGSVK PYTNFDAE SEQ ID NO: 46:RKKRRQRRRRVKRISQAVHAAHAEINEAGRRVKRKVPRNQDWLRVKRASFEAQGALANIAVDKARVKRSIINFEKLRVKRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 69:KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEGSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Arrangement of Components a), b), and c) in the Complex for UseAccording to the Present Invention

The components a), b), and c) may be arranged in the complex for useaccording to the present invention in any way.

In particular if more than one cell penetrating peptide and/or more thanone antigen or antigenic epitope and/or more than one TLR peptideagonist are comprised by the complex for use according to the presentinvention, the more than one cell penetrating peptide may be positionedin a non-consecutive manner, i.e. at least one antigen or antigenicepitope (component b)) and/or at least one TLR peptide agonist(component c)) may interrupt a stretch of consecutively positioned cellpenetrating peptides and/or the cell penetrating peptides may bepositioned with component b) and/or with component c) in an alternatingmanner. Similarly, the more than one antigen or antigenic epitope may bepositioned in a non-consecutive manner, i.e. at least one cellpenetrating peptide (component a)) and/or at least one TLR peptideagonist (component c)) may interrupt a stretch of consecutivelypositioned antigens or antigenic epitopes and/or the antigens orantigenic epitopes may be positioned with component a) and/or withcomponent c) in an alternating manner. Similarly, the more than one TLRpeptide agonist may be positioned in a non-consecutive manner, i.e. atleast one cell penetrating peptide (component a)) and/or at least oneantigen or antigenic epitope (component b)) may interrupt a stretch ofconsecutively positioned TLR peptide agonists and/or the TLR peptideagonists may be positioned with component a) and/or with component b) inan alternating manner.

However, it is preferred that the more than one cell penetrating peptideis positioned in the complex for use according to the present inventionin a consecutive manner and/or the more than one antigen or antigenicepitope is positioned in the complex for use according to the presentinvention in a consecutive manner and/or the more than one TLR peptideagonist is positioned in the complex for use according to the presentinvention in a consecutive manner. This means in particular that allsingle units of a certain component, i.e. all cell penetrating peptides,all antigens or antigenic epitopes or all TLR peptide agonists, whichare comprised by the complex are positioned in a stretch, which is notinterrupted by any of the other two components. Rather, the other twocomponents are positioned in the complex for example before or aftersuch a stretch of all single units of said certain component. However,the single units of said certain component positioned consecutively insuch a way may be linked to each other for example by a spacer or linkeras described herein, which is not of the other two components.

It is particularly preferred that each of the components a), b), and c)is positioned in a consecutive manner.

Structurally each component a), b), and c) typically comprises a singlemain chain and at least one side chain. The term “main chain” (also“backbone chain”), as used in the context of the present invention,refers to the main continuous chain of covalently bond atoms in amolecule. For example, in peptides, polypeptides and proteins, the mainchain (backbone) typically comprises alpha-carbon atoms and nitrogenatoms of the constituent amino acids linked by the peptide bond. Thebackbone does not include the side chains. The term “side chain” (also“pendant chain”), as used in the context of the present invention,refers to a chemical group that is attached to a core part of themolecule called “main chain” or backbone. For example, in peptides,polypeptides and proteins, the side chains typically represent the(main) parts of the constituent amino acids, which are attached to thealpha-carbon atoms of the backbone.

In the complex for use according to the present invention, thecomponents a), b), and c) may be covalently linked via a linker orspacer as described herein or they may be directly covalently linked.Independently of whether a spacer or linker is used for covalent linkageor not, there are in principle four options of how two of the threecomponents are linked to each other in the complex for use according tothe present invention, namely:

-   -   (i) via main-chain/main-chain linkage,    -   (ii) via main-chain/side-chain linkage,    -   (iii) via side-chain/main-chain linkage or    -   (iv) via side-chain/side chain linkage.

Preferably, all three components a), b), and c) are linked viamain-chain/main-chain linkage, thus resulting in particular in a mainchain of the complex for use according to the present invention, whichcomprises the main chain of one or more cell penetrating peptide(s), themain chain of one or more antigen(s) or antigenic epitope(s), and themain chain of one or more TLR peptide agonist(s). In other words, themain chain of one or more cell penetrating peptide(s), the main chain ofone or more antigen(s) or antigenic epitope(s), and the main chain ofone or more TLR peptide agonist(s) constitute the main chain of thecomplex for use according to the present invention, optionally togetherwith further components, for example linker(s), spacer(s), etc. . . .Accordingly, the following arrangements of the components a), b), and c)are preferred, in particular if the at least one antigen or antigenicepitope is a peptide, polypeptide or protein, whereby said preferredarrangements are shown below in N-terminus→C-terminus direction of themain chain of the complex and wherein all three components a), b), andc) are linked via main-chain/main-chain linkage and may be optionallylinked by a linker, a spacer or another additional component:

-   -   (α) component a) (cell penetrating peptide)—component b) (at        least one antigen or antigenic epitope)—component c) (at least        one TLR peptide agonist);    -   (β) component c) (at least one TLR peptide agonist)—component a)        (cell penetrating peptide)—component b) (at least one antigen or        antigenic epitope);    -   (γ) component a) (cell penetrating peptide)—component c) (at        least one TLR peptide agonist)—component b) (at least one        antigen or antigenic epitope);    -   (δ) component c) (at least one TLR peptide agonist)—component b)        (at least one antigen or antigenic epitope)—component a) (cell        penetrating peptide);    -   (ε) component b) (at least one antigen or antigenic        epitope)—component a) (cell penetrating peptide)—component c)        (at least one TLR peptide agonist); or    -   (ζ) component b) (at least one antigen or antigenic        epitope)—component c) (at least one TLR peptide        agonist)—component a) (cell penetrating peptide).

In particular if all three components a), b), and c) are linked viamain-chain/main-chain linkage, it is preferred that the at least oneantigen or antigenic epitope is positioned C-terminally of the cellpenetrating peptide, whereby the cell penetrating peptide and the atleast one antigen or antigenic epitope are optionally linked by afurther component, e.g. a linker, a spacer, or by the at least one TLRpeptide agonist. Accordingly, this corresponds to the arrangements (α),(β), and (γ) from the arrangements shown above, i.e. from the abovearrangements arrangements (α), (β), and (γ) are more preferred.

Even more preferably, the at least one antigen or antigenic epitope ispositioned C-terminally of the cell penetrating peptide, whereby thecell penetrating peptide and the at least one antigen or antigenicepitope are optionally linked by a further component, e.g. a linker, aspacer, but not by the at least one TLR peptide agonist. Accordingly,this corresponds to the arrangements (α) and (β) from the arrangementsshown above, i.e. from the above arrangements arrangements (α) and (β)are even more preferred. Particularly preferably, the complex for useaccording to the present invention is a recombinant polypeptide or arecombinant protein and the components a) to c) are positioned inN-terminus→C-terminus direction of the main chain of said complex in theorder:

(α) component a)—component b)—component c); or

(β) component c)—component a)—component b),

wherein the components may be linked by a further component, inparticular by a linker or a spacer.

Particularly preferred is arrangement (a), wherein the at least one TLRagonist comprises or consists of at least one TLR2 agonist, for example:

-   -   (α1) component a) (cell penetrating peptide)—component b) (at        least one antigen or antigenic epitope)—one or more TLR2 peptide        agonist, e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s);    -   (α2) component a) (cell penetrating peptide)—component b) (at        least one antigen or antigenic epitope)—one or more TLR2 peptide        agonist, e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s), one or        more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4 peptide        agonist(s) and one or more TLR5 peptide agonist, e.g. 1, 2, 3,        4, or 5 TLR5 peptide agonist(s);    -   (α3) component a) (cell penetrating peptide)—component b) (at        least one antigen or antigenic epitope)—one or more TLR2 peptide        agonist, e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s) and one        or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4 peptide        agonist(s); or    -   (α4) component a) (cell penetrating peptide)—component b) (at        least one antigen or antigenic epitope)—one or more TLR2 peptide        agonist, e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s) and one        or more TLR5 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR5 peptide        agonist(s).

Alternatively, in such an arrangement comprising a TLR2 peptide agonist,additional TLR peptide agonists may also be arranged at other positionsin the complex, for example:

-   -   (α5) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s)—component a) (cell penetrating        peptide)—component b) (at least one antigen or antigenic        epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR2 peptide agonist(s);    -   (α6) one or more TLR5 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR5 peptide agonist(s)—component a) (cell penetrating        peptide)—component b) (at least one antigen or antigenic        epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR2 peptide agonist(s); or    -   (α7) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s) and one or more TLR5 peptide agonist,        e.g. 1, 2, 3, 4, or 5 TLR5 peptide agonist(s)—component a) (cell        penetrating peptide)—component b) (at least one antigen or        antigenic epitope)—one or more TLR2 peptide agonist, e.g. 1, 2,        3, 4, or 5 TLR2 peptide agonist(s).

Particularly preferred is arrangement (β), wherein the at least one TLRagonist comprises or consists of at least one TLR4 agonist, for example:

-   -   (β1) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s)—component a) (cell penetrating        peptide)—component b) (at least one antigen or antigenic        epitope);    -   (β2) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s), one or more TLR2 peptide agonist, e.g.        1, 2, 3, 4, or 5 TLR2 peptide agonist(s) and one or more TLR5        peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR5 peptide        agonist(s)—component a) (cell penetrating peptide)—component b)        (at least one antigen or antigenic epitope);    -   (β3) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s) and one or more TLR2 peptide agonist,        e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s)—component a) (cell        penetrating peptide)—component b) (at least one antigen or        antigenic epitope); or    -   (β4) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s) and one or more TLR5 peptide agonist,        e.g. 1, 2, 3, 4, or 5 TLR5 peptide agonist(s)—component a) (cell        penetrating peptide)—component b) (at least one antigen or        antigenic epitope).

Alternatively, in such an arrangement comprising a TLR4 peptide agonist,additional TLR peptide agonists may also be arranged at other positionsin the complex, for example:

-   -   (β5) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s)—component a) (cell penetrating        peptide)—component b) (at least one antigen or antigenic        epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR2 peptide agonist(s);    -   (β6) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s)—component a) (cell penetrating        peptide)—component b) (at least one antigen or antigenic        epitope)—one or more TLR5 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR5 peptide agonist(s); or    -   (β7) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR4 peptide agonist(s)—component a) (cell penetrating        peptide)—component b) (at least one antigen or antigenic        epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5        TLR2 peptide agonist(s) and one or more TLR5 peptide agonist,        e.g. 1, 2, 3, 4, or 5 TLR5 peptide agonist(s).

Alternatively, only two of the three components a), b), and c) arelinked via main-chain/main-chain linkage in the complex for useaccording to the present invention.

For example components a) and b) are linked via main-chain/main-chainlinkage, resulting thus in the following arrangements of the componentsa) and b) in the complex, shown in N-terminus→C-terminus direction ofthe main chain of the complex, whereby the components a) and b) may beoptionally linked by a further component, e.g. a linker, a spacer etc.:

-   -   (1) cell penetrating peptide (a)—antigen/antigenic epitope (b);        or    -   (2) antigen/antigenic epitope (b)—cell penetrating peptide (a).        In such a case, component c), i.e. the at least one TLR peptide        agonist, may then be arranged via main-chain/side-chain linkage,        via side-chain/main-chain linkage or via side-chain/side chain        linkage to either the cell penetrating peptide (a) or to the        antigen/antigenic epitope (b) or, if present, to an additional        component like a spacer or linker, which may be, for example,        positioned between the cell penetrating peptide (a) and the        antigen/antigenic epitope (b). This includes the following        arrangements:    -   (i) component c) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component a), i.e.        the main chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        side chain of the cell penetrating peptide;    -   (ii) component c) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component a), i.e.        the side chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        main chain of the cell penetrating peptide;    -   (iii) component c) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component a), i.e.        the side chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        side chain of the cell penetrating peptide;    -   (iv) component c) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component b), i.e.        the main chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one antigen or antigenic epitope;    -   (v) component c) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component b), i.e.        the side chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        main chain of the at least one antigen or antigenic epitope;    -   (vi) component c) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component b), i.e.        the side chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one antigen or antigenic epitope;    -   (vii)component c) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to a linker or a spacer        positioned between component a) and component b), i.e. the main        chain of the at least one TLR peptide agonist is covalently        linked—optionally via a spacer or a linker—to the side chain of        a linker or a spacer positioned between component a) and        component b);    -   (viii) component c) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to a linker or a spacer        positioned between component a) and component b), i.e. the side        chain of the at least one TLR peptide agonist is covalently        linked —optionally via a spacer or a linker—to the main chain of        a linker or a spacer positioned between component a) and        component b); or    -   (ix) component c) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to a linker or a spacer        positioned between component a) and component b), i.e. the side        chain of the at least one TLR peptide agonist is covalently        linked—optionally via a spacer or a linker—to the side chain of        a linker or a spacer positioned between component a) and        component b).

For example components b) and c) are linked via main-chain/main-chainlinkage, resulting thus in the following arrangements of the componentsb) and c) in the complex, shown in N-terminus→C-terminus direction ofthe main chain of the complex, whereby the components b) and c) may beoptionally linked by a further component, e.g. a linker, a spacer etc.:

-   -   (3) antigen/antigenic epitope (b)—TLR peptide agonist (c); or    -   (4) TLR peptide agonist (c)—antigen/antigenic epitope (b).

In such a case, component a), i.e. the cell penetrating peptide, maythen be arranged via main-chain/side-chain linkage, viaside-chain/main-chain linkage or via side-chain/side chain linkage toeither the antigen/antigenic epitope (b) or to the TLR peptide agonist(c) or, if present, to an additional component like a spacer or linker,which may be, for example, positioned between the antigen/antigenicepitope (b) and the TLR peptide agonist (c). This includes the followingarrangements:

-   -   (x) component a) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component b), i.e.        the main chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        the at least one antigen or antigenic epitope;    -   (xi) component a) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component b), i.e.        the side chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the main chain of        the at least one antigen or antigenic epitope;    -   (xii) component a) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component b), i.e.        the side chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        the at least one antigen or antigenic epitope;    -   (xiii) component a) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component c), i.e.        the main chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        the at least one TLR peptide agonist;    -   (xiv) component a) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component c), i.e.        the side chain the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the main chain of        the at least one TLR peptide agonist;    -   (xv)component a) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain/side-chain linkage to component        c), i.e. the side chain of the cell penetrating peptide is        covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one TLR peptide agonist;    -   (xvi) component a) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to a linker or a spacer        positioned between component b) and component c), i.e. the main        chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        a linker or a spacer positioned between component b) and        component c);    -   (xvii) component a) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to a linker or a spacer        positioned between component b) and component c), i.e. the side        chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the main chain of        a linker or a spacer positioned between component b) and        component c); or    -   (xviii) component a) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to a linker or a spacer        positioned between component b) and component c), i.e. the side        chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        a linker or a spacer positioned between component b) and        component c).

For example components a) and c) are linked via main-chain/main-chainlinkage, resulting thus in the following arrangements of the componentsa) and b) in the complex, shown in N-terminus→C-terminus direction ofthe main chain of the complex, whereby the components a) and c) may beoptionally linked by a further component, e.g. a linker, a spacer etc.:

-   -   (5) cell penetrating peptide (a)—TLR peptide agonist (c); or    -   (6) TLR peptide agonist (c)—cell penetrating peptide (a).

In such a case, component b), i.e. the at least one antigen or antigenicepitope, may then be arranged via main-chain/side-chain linkage, viaside-chain/main-chain linkage or via side-chain/side chain linkage toeither the cell penetrating peptide (a) or to the TLR peptide agonist(c) or, if present, to an additional component like a spacer or linker,which may be, for example, positioned between the cell penetratingpeptide (a) and the TLR peptide agonist (c). This includes the followingarrangements:

-   -   (xix) component b) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component a), i.e.        the main chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        side chain of the cell penetrating peptide;    -   (xx) component b) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component a), i.e.        the side chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        main chain of the cell penetrating peptide;    -   (xxi) component b) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component a), i.e.        the side chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        side chain of the cell penetrating peptide;    -   (xxii) component b) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component c), i.e.        the main chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one TLR peptide agonist;    -   (xxiii) component b) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component c), i.e.        the side chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        main chain of the at least one TLR peptide agonist;    -   (xxiv) component b) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component c), i.e.        the side chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one TLR peptide agonist;    -   (xxv) component b) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to a linker or a spacer        positioned between component a) and component c), i.e. the main        chain of the at least one antigen or antigenic epitope is        covalently linked—optionally via a spacer or a linker—to the        side chain of a linker or a spacer positioned between        component a) and component c);    -   (xxvi) component b) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to a linker or a spacer        positioned between component a) and component c), i.e. the side        chain of the at least one antigen or antigenic epitope is        covalently linked—optionally via a spacer or a linker—to the        main chain of a linker or a spacer positioned between        component a) and component c); or    -   (xxvii) component b) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to a linker or a spacer        positioned between component a) and component c), i.e. the side        chain of the at least one antigen or antigenic epitope is        covalently linked—optionally via a spacer or a linker—to the        side chain of a linker or a spacer positioned between        component a) and component c).

Alternatively, it is also conceivable that in the complex for useaccording to the present invention all three of the components a), b),and c) are arranged via main-chain/side-chain linkage, viaside-chain/main-chain linkage or via side-chain/side chainlinkage,optionally linked by an additional component, e.g. a spacer or a linker.

Colorectal Cancer

The present invention provides the complex as described above for use inthe prevention and/or treatment of colorectal cancer.

Colorectal cancer (CRC, also known as “bowel cancer”) is a cancer thatcomprises colon cancers and rectal cancers (CC). Both individual cancershave many features in common, but the cancer starting point. Accordingto Siegel, R., C. Desantis, and A. Jemal, Colorectal cancer statistics,2014. CA Cancer J Clin, 2014. 64(2): p. 104-17, in the United Statesbetween 2006 and 2010, the incidence by tumor site is slightly moreimportant in the proximal colon (first and middle parts of the colon).With about 19 cases on 100,000 people, it represents 42% of the cases.It is followed by the rectal cancer, with 28% of the cases and thedistal colon (bottom part of the colon) with an incidence of 10 cases on100,000 people.

Anatomically, the term “colorectal cancer” includes (i) cancers ofcolon, such as cancers of cecum (including cancers the ileocecal valve),appendix, ascending colon, hepatic flexure, transverse colon, splenicflexure, descending colon, sigmoid colon (including cancers of sigmoid(flexure)) as well as cancers of overlapping sites of colon; (ii)cancers of rectosigmoid junction, such as cancers of colon and rectumand cancers of rectosigmoid; and (iii) cancers of rectum, such ascancers of rectal ampulla.

Preferably, the colorectal cancer is a cancer of colon, such as a cancerof cecum (including cancer the ileocecal valve), cancer of appendix,cancer of ascending colon, cancer of hepatic flexure, cancer oftransverse colon, cancer of splenic flexure, cancer of descending colon,cancer of sigmoid colon (including cancers of sigmoid (flexure)) or acombination thereof.

It is also preferred that the colorectal cancer is a cancer ofrectosigmoid junction, such as (i) a cancer of colon and rectum or (ii)a cancer of rectosigmoid.

Furthermore, it is also preferred that the colorectal cancer is a cancerof rectum, such as a cancer of rectal ampulla.

Regarding the cell type, colorectal cancers include colorectaladenocarcinoma, colorectal stromal tumors, primary colorectal lymphoma,colorectal leiomyosarcoma, colorectal melanoma, colorectal squamous cellcarcinoma and colorectal carcinoid tumors, such as, for example,carcinoid tumors of cecum, appendix, ascending colon, transverse colon,descending colon, sigmoid colon and/or rectum. Thus, preferred types ofcolorectal cancers include colorectal adenocarcinoma, colorectal stromaltumors, primary colorectal lymphoma, colorectal leiomyosarcoma,colorectal melanoma, colorectal squamous cell carcinoma and colorectalcarcinoid tumors, such as, for example, carcinoid tumors of cecum,appendix, ascending colon, transverse colon, descending colon, sigmoidcolon and/or rectum. More preferably, the colorectal cancer is acolorectal adenocarcinoma or a colorectal carcinoid carcinoma. Even morepreferably, the colorectal cancer is a colorectal adenocarcinoma.

More than 95% of CRCs are adenocarcinomas. Colorectal adenocarcinomastypically start from glandular cells that make mucus to lubricate thecolon or rectum. CRC typically starts in the innermost layer and cangrow through some or all of the other layers. In rare cases, CRC couldform in a polyp, which facilitates its growth into the wall of startingregion. In advanced stage (stage III and IV), the cancer travels tonearby lymph nodes or to distant parts of the body through bloodvessels.

For example, in colorectal cancer, the TNM staging system includes thefollowing stages for primary tumors (“T” stages): TX—Primary tumourcannot be assessed, T0—No evidence of primary tumour, Ta—Non-invasivepapillary carcinoma, Tis—Carcinoma in situ: intraepithelial or invasionof lamina propria, T1—Tumour invades submucosa, T2—Tumour invadesmuscularis propria, T3—Tumour invades through the muscularis propriainto the pericolorectal tissues, T4a—Tumour penetrates to the surface ofthe visceral peritoneum and T4b—Tumour directly invades or is adherentto other organs or structures; following stages for lymph nodes (“N”stages): NX—Regional lymph nodes cannot be assessed, N0—No regionallymph node metastasis, N1—Metastasis in 1-3 regional lymph nodes withN1a—Metastasis in 1 regional lymph node, N1b—Metastasis in 2-3 regionallymph nodes and N1c—Tumor deposit(s) in the subserosa, mesentery, ornonperitonealized pericolic or perirectal tissues without regional nodalmetastasis, N2—Metastasis in 4 or more lymph nodes with N2a—Metastasisin 4-6 regional lymph nodes and N2b—Metastasis in 7 or more regionallymph nodes; and the following stages for distant metastasis (“M”stages): M0—No distant metastasis and M1—Distant metastasis withM1a—Metastasis confined to 1 organ or site (eg, liver, lung, ovary,nonregional node) and M1b—Metastases in more than 1 organ/site or theperitoneum.

This stages can be integrated into the following numerical staging ofcolorectal cancer: Stage 0: Tis, N0, M0; Stage I: T1, N0, M0 or T2, N0,M0; Stage IIA: T3, N0, M0; Stage IIB: T4a, N0, M0; Stage IIC: T4b, N0,M0; Stage IIIA: T1-T2, N1/N1c, M0 or T1, N2a, M0; Stage IIIB: T3-T4a,N1/N1c, M0 or T2-T3, N2a, M0 or T1-T2, N2b, M0; Stage IIIC: T4a, N2a, M0or T3-T4a, N2b, M0 or T4b, N1-N2, M0; Stage IVA: any T, any N, M1a andStage IVB: any T, any N, M1b. Briefly, in Stage 0, the cancer has notgrown beyond the inner layer of the colon or rectum; in Stage I thecancer has spread from the mucosa to the muscle layer; in Stage II thecancer has spread through the muscle layer to the serosa nearby organs;in Stage III the cancer has spread to nearby lymph node(s) or cancercells have spread to tissues near the lymph nodes; and in Stage IV thecancer has spread through the blood and lymph nodes to other parts ofthe body.

Despite the term “cancer”, colorectal cancer includes all numericalstages as described above, and, thus, a preferred stage of colorectalcancer may be selected from the group consisting of Stage 0 (Tis, N0,M0), Stage I (T1, N0, M0 or T2, N0, M0), Stage IIA (T3, N0, M0), StageIIB (T4a, N0, M0), Stage IIC (T4b, N0, M0), Stage IIIA (T1-T2, N1/N1c,M0 or T1, N2a, M0), Stage IIIB (T3-T4a, N1/N1c, M0 or T2-T3, N2a, M0 orT1-T2, N2b, M0), Stage IIIC (T4a, N2a, M0 or T3-T4a, N2b, M0 or T4b,N1-N2, M0), Stage IVA (any T, any N, M1a) and Stage IVB (any T, any N,M1b). More preferably, the colorectal cancer is selected from the groupconsisting of Stage I (T1, N0, M0 or T2, N0, M0), Stage IIA (T3, N0,M0), Stage IIB (T4a, N0, M0), Stage IIC (T4b, N0, M0), Stage IIIA(T1-T2, N1/N1c, M0 or T1, N2a, M0), Stage IIIB (T3-T4a, N1/N1c, M0 orT2-T3, N2a, M0 or T1-T2, N2b, M0), Stage IIIC (T4a, N2a, M0 or T3-T4a,N2b, M0 or T4b, N1-N2, M0), Stage IVA (any T, any N, M1a) and Stage IVB(any T, any N, M1b). Even more preferably, the colorectal cancer isselected from the group consisting of Stage IIA (T3, N0, M0), Stage IIB(T4a, N0, M0), Stage IIC (T4b, N0, M0), Stage IIIA (T1-T2, N1/N1c, M0 orT1, N2a, M0), Stage IIIB (T3-T4a, N1/N1c, M0 or T2-T3, N2a, M0 or T1-T2,N2b, M0), Stage IIIC (T4a, N2a, M0 or T3-T4a, N2b, M0 or T4b, N1-N2,M0), Stage IVA (any T, any N, M1a) and Stage IVB (any T, any N, M1b).Most preferably, the colorectal cancer is (i) Stage III colorectalcancer, such as Stage IIIA (T1-T2, N1/N1c, M0 or T1, N2a, M0), StageIIIB (T3-T4a, N1/N1c, M0 or T2-T3, N2a, M0 or T1-T2, N2b, M0), or StageIIIC (T4a, N2a, M0 or T3-T4a, N2b, M0 or T4b, N1-N2, M0), or (ii) StageIV colorectal cancer, such as Stage IVA (any T, any N, M1a) and StageIVB (any T, any N, M1b).

Nucleic Acid Encoding the Peptides and Protein Complexes

In another aspect the present invention provides a nucleic acid encodingthe complex as described herein, wherein the complex is a polypeptide ora protein, for use in the prevention and/or treatment of colorectalcancer. In particular, the present invention provides polynucleotidesfor use in the prevention and/or treatment of colorectal cancer, saidpolynucleotides encoding the complex as defined above.

In this context, nucleic acids preferably comprise single stranded,double stranded or partially double stranded nucleic acids, preferablyselected from genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisenseRNA, ribozyme, complimentary RNA/DNA sequences with or withoutexpression elements, a mini-gene, gene fragments, regulatory elements,promoters, and combinations thereof.

Preferably, the invention relates to a nucleic acid for use according tothe present invention, said nucleic acid encoding a complex, which is inparticular a polypeptide or protein, said complex comprising a cellpenetrating peptide, at least one antigen or antigenic epitope, which isa polypeptide or protein, and at least one TLR peptide agonist, whereinthe cell penetrating peptide, the at least one antigen or antigenicepitope, and the at least one TLR peptide agonist are covalently linked,optionally with peptidic spacer(s) or linker(s) as described herein. Ifmore than one antigen or antigenic epitope, which is a polypeptide orprotein, is comprised by said complex, the more than one antigens orantigenic epitopes are also covalently linked, optionally with peptidicspacer(s) or linker(s) as described herein. Similarly, if more than oneTLR peptide agonist is comprised by said complex, the more than one TLRpeptide agonists are also covalently linked, optionally with peptidicspacer(s) or linker(s) as described herein.

Particularly preferably the nucleic acid for use according to thepresent invention encodes a complex which is a (recombinant) fusionprotein comprising (a) a cell penetrating peptide as described above,(b) at least one, preferably at least two, more preferably at leastthree, even more preferably at least four, particularly preferably atleast five, most preferably at least six antigens or antigenic epitopesas described above, preferably arranged in a consecutive manner asdescribed above and (c) at least one TLR agonist as described above.

Production and Purification of the Complexes

According to a further aspect the present invention provides a vectorfor use in the prevention and/or treatment of colorectal cancer, inparticular a recombinant vector, comprising a nucleic acid as describedabove.

The term “vector”, as used in the context of the present invention,refers to a nucleic acid molecule, preferably to an artificial nucleicacid molecule, i.e. a nucleic acid molecule which does not occur innature. A vector in the context of the present invention is suitable forincorporating or harboring a desired nucleic acid sequence. Such vectorsmay be storage vectors, expression vectors, cloning vectors, transfervectors etc. A storage vector is a vector which allows the convenientstorage of a nucleic acid molecule. Thus, the vector may comprise asequence corresponding, e.g., to a desired antibody or antibody fragmentthereof according to the present invention. An expression vector may beused for production of expression products such as RNA, e.g. mRNA, orpeptides, polypeptides or proteins. For example, an expression vectormay comprise sequences needed for transcription of a sequence stretch ofthe vector, such as a promoter sequence. A cloning vector is typically avector that contains a cloning site, which may be used to incorporatenucleic acid sequences into the vector. A cloning vector may be, e.g., aplasmid vector or a bacteriophage vector. A transfer vector may be avector which is suitable for transferring nucleic acid molecules intocells or organisms, for example, viral vectors. A vector in the contextof the present invention may be, e.g., an RNA vector or a DNA vector.Preferably, a vector is a DNA molecule. For example, a vector in thesense of the present application comprises a cloning site, a selectionmarker, such as an antibiotic resistance factor, and a sequence suitablefor multiplication of the vector, such as an origin of replication.Preferably, a vector in the context of the present application is aplasmid vector. Preferably, a vector in the context of the presentapplication is an expression vector.

Cells transformed with a vector as described above for use in theprevention and/or treatment of colorectal cancer are also includedwithin the scope of the invention. Examples of such cells include, butare not limited to, bacterial cells, e.g. E. coli, and eukaryotic cells,e.g., yeast cells, animal cells or plant cells. In one embodiment thecells are mammalian, e.g., human, CHO, HEK293T, PER.C6, NS0, myeloma orhybridoma cells. Accordingly, the present invention also relates to acell expressing the antibody, or the antigen binding fragment thereof,for use according to the present invention; or comprising the vector foruse according to the present invention.

In particular, a cell may be transfected with a vector as describedabove, preferably with an expression vector. The term “transfection”refers to the introduction of nucleic acid molecules, such as DNA or RNA(e.g. mRNA) molecules, into cells, preferably into eukaryotic cells. Inthe context of the present invention, the term “transfection”encompasses any method known to the skilled person for introducingnucleic acid molecules into cells, preferably into eukaryotic cells,such as into mammalian cells. Such methods encompass, for example,electroporation, lipofection, e.g. based on cationic lipids and/orliposomes, calcium phosphate precipitation, nanoparticle basedtransfection, virus based transfection, or transfection based oncationic polymers, such as DEAE-dextran or polyethylenimme etc.Preferably, the introduction is non-viral.

Numerous expression systems can be used, including without limitationchromosomes, episomes, and derived viruses. More particularly, thevector as described above, in particular the recombinant vector used,can be derived from bacterial plasmids, transposons, yeast episomes,insertion elements, yeast chromosome elements, viruses such asbaculovirus, papilloma viruses such as SV40, vaccinia viruses,adenoviruses, fox pox viruses, pseudorabies viruses, retroviruses.

For example, such vectors, in particular recombinant vectors, canequally be cosmid or phagemid derivatives. The nucleotide sequence, inparticular the nucleic acid according to the present invention, may beinserted in the recombinant expression vector by methods well known to aperson skilled in the art such as, for example, those described inMOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 4th Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.

The vector, in particular the recombinant vector, may also includenucleotide sequences that control the regulation of the expression, inparticular of the nucleic acid for use according to the presentinvention, as well as nucleotide sequences permitting the expression,the transcription, and the translation, in particular of the nucleicacid for use according to the present invention. Typically, thesesequences are selected according to the host cells used.

Thus, for example, an appropriate secretion signal can be integrated inthe vector for use according to the present invention, in particular ina recombinant vector, so that the polypeptide or protein encoded by thenucleic acid for use according to the present invention, will bedirected, for example towards the lumen of the endoplasmic reticulum,towards the periplasmic space, on the membrane or towards theextracellular environment. The choice of an appropriate secretion signalmay facilitate subsequent protein purification.

In yet another aspect the present invention provides a host cell for usein the prevention and/or treatment of colorectal cancer, the host cellcomprising a vector, in particular a recombinant vector, as describedherein.

The introduction of the vector, in particular the recombinant vector,into a host cell can be carried out according to methods that are wellknown to a person skilled in the art, such as those described in BASICMETHODS IN MOLECULAR BIOLOGY, Davis et al., 2nd ed., McGraw-HillProfessional Publishing, 1995, and MOLECULAR CLONING: A LABORATORYMANUAL, supra, including for example transfection as described above,e.g. by calcium phosphate, by DEAE dextran, or by cationic lipids;microinjection, electroporation, transduction or infection.

The host cell can be, for example, bacterial cells such as E. coli,cells of fungi such as yeast cells and cells of Aspergillus,Streptomyces, insect cells, and/or any cell line, e.g. Chinese HamsterOvary cells (CHO), C127 mouse cell line, BHK cell line of Syrian hamstercells, Human Embryonic Kidney 293 (HEK 293) cells. Preferably, the hostcell for use according to the present invention is mammalian, e.g.,human, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Dendriticcells and dendritic cell lines are particularly preferred as a hostcell. Typically, the choice of a culture medium depends in particular onthe choice of the cell type and/or the cell line, whereby the skilledperson is aware of suitable culture media, which are appropriate for aselected cell type and/or cell line.

The host cells can be used, for example, to express a polypeptide orprotein, in particular the complex for use according to the presentinvention, on the basis of the vector and/or the nucleic acid asdescribed herein. After purification by standard methods, the expressedpolypeptide or protein, in particular the complex for use according tothe present invention, can be used as described herein.

Accordingly, the present invention also provides a method for preparingthe complex as defined herein, in particular wherein the complex is apolypeptide or protein. Said method comprises the steps of:

-   -   (i) cultivating a host cell as described above in a culture        medium; and    -   (ii) separating the complex as defined herein from the culture        medium or separating the complex as defined herein from the host        cell lysate after host cell lysis.

Thus, the complex obtained by such a method according to the presentinvention is preferably a complex for use according to the presentinvention as described herein.

For protein extraction commercially available kits and/or reagents maybe used, for example BugBuster™ from Novagen.

Preferably, the method for preparing the complex as defined hereinfurther comprises the following step:

-   -   (iii) solubilization of the complex as defined herein, e.g. by        resuspension in solutions containing urea or guanidine        hydrochloride (GuHCl),

wherein step (iii) follows step (ii) as described above.

Moreover, it is preferred that the method for preparing the complex asdefined herein further comprises the following step:

-   -   (iv) purification of the complex as defined herein, preferably        by one-step affinity chromatography,

wherein step (iv) follows step (ii), or, if present, step (iii) asdescribed above.

In addition, the complex as defined herein may also be prepared bysynthetic chemistry methods, for example by solid-phase peptidesynthesis.

Purification of those peptides or proteins may be carried out by meansof any technique known in the art for protein/peptide purification.Exemplary techniques include ion-exchange chromatography, hydrophobicinteraction chromatography, and immunoaffinity methods.

Thus, the present invention also provides a method for preparing thecomplex as defined herein comprising the steps of:

-   -   (i) chemically synthesizing said complex; and    -   (ii) purifying said complex.

Preferably, in the method for preparing a complex as defined herein, thecomplex chemically synthesized in step (i) and purified in step (ii)comprises an amino acid sequences as described herein for a cellpenetrating peptide, an amino acid sequence as described herein for aTLR peptide agonist, and, optionally if the at least one antigen and/orantigenic epitope is a peptide or a protein, an amino acid sequence asdescribed herein for an antigen or antigenic epitope.

Alternatively, the present invention also provides a method forpreparing the complex as defined herein, wherein

-   -   (i) the cell penetrating peptide, the at least one antigen or        antigenic fragment and/or the at least one TLR peptide agonist        are synthesized separately;    -   (ii) optionally, the cell penetrating peptide, the at least one        antigen or antigenic fragment and/or the at least one TLR        peptide agonist are purified; and    -   (iii) the cell penetrating peptide, the at least one antigen or        antigenic fragment and/or the at least one TLR peptide agonist        are covalently linked as described above, optionally by a spacer        or linker or by a cross-linking agent as described above.

Cells Loaded with the Complexes According to the Invention

In yet another aspect the present invention relates to a cell loadedwith the complex as defined herein for use in the prevention and/ortreatment of colorectal cancer. For example, the cells loaded with thecomplex as defined herein are cells from a subject to be treated, inparticular isolated cells from a subject to be treated, i.e. cellsisolated from a subject to be treated.

As used in the context of the present invention, the term “subject”refers in particular to mammals. For example, mammals contemplated bythe present invention include human, primates, domesticated animals suchas cattle, sheep, pigs, horses, laboratory rodents and the like. Morepreferably, the term “subject” refers to a human subject.

As used in the context of the present invention, “treatment” and“treating” and the like generally mean obtaining a desiredpharmacological and physiological effect. The effect may be prophylacticin terms of preventing or partially preventing a disease, a symptom or acondition thereof and/or may be therapeutic in terms of a partial orcomplete cure of a disease, a condition, a symptom or an adverse effectattributed to the disease. The term “treatment” as used herein coversany treatment of a disease in a mammal, in particular in a human, andincludes: (a) preventing the disease from occurring in a subject who maybe predisposed to the disease but the outbreak of the disease has notyet occurred and/or the disease has not yet been diagnosed in thissubject, for example a preventive early asymptomatic intervention; (b)inhibiting the disease, i.e., arresting or slowing down its development;or (c) relieving the disease, i.e., causing an at least partialregression of the disease and/or of at least one of its symptoms orconditions such as improvement or remediation of damage. In particular,the methods, uses, formulations and compositions according to theinvention are useful in the treatment of cancers or infectious diseasesand/or in the prevention of evolution of cancers into an advanced ormetastatic stage in subjects with early stage cancer, thereby improvingthe staging of the cancer. When applied to cancers, prevention of adisease or disorder includes the prevention of the appearance ordevelopment of a cancer in an individual identified as at risk ofdeveloping said cancer, for instance due to past occurrence of saidcancer in the circle of the individual's relatives, and prevention ofinfection with tumor promoting pathogens such as, for example,Epstein-Barr virus (EBV), Human papillomavirus (HPV), Hepatitis B virus(HBV), Hepatitis C virus (HCV), Human Herpes virus 8 (HHV8), humanT-cell leukemia virus type 1 (HTLV-1), Merkel cell polyomavirus (MCV)and Helicobacter pylori. Also covered by the terms“prevention/treatment” of a cancer is the stabilization or delay of analready diagnosed cancer in an individual. By “stabilization”, it ismeant the prevention of evolution of cancer into advanced or metastaticstage in subjects with early stage cancer.

Preferably, the cell loaded with the complex as defined herein is anantigen-presenting cell (APC). Preferably, the antigen presenting cellis selected from the group consisting of a dendritic cell (DC), amacrophage and a B-cell. Dendritic cells, in particular dendritic cells(conventional and/or plasmacytoid) isolated from a subject to betreated, are more preferred.

Methods to isolate antigen-presenting cells, in particular dendriticcells, from a subject are known to the skilled person. They includeharvesting monocytes or hematopoietic stem cells from bone marrow, cordblood, or peripheral blood. They also include the use of embryonic stem(ES) cells and induced pluripotent stem cells (iPS). Antigen presentingcells, in particular dendritic cells or their precursors, can beenriched by methods including elutriation and magnetic bead basedseparation, which may involve enrichment for CD14+ precursor cells.

Methods to load the complex as defined herein into the cells, preferablyinto the above-mentioned antigen presenting cells, more preferably intodendritic cells, and further to prepare such cells before administrationto a subject are known to one skilled in the art. For example,preparation of dendritic cells can include their culture ordifferentiation using cytokines that may include for example GM-CSF andIL-4. Dendritic cell lines may also be employed. Loading of the complexof the invention into the cells, preferably into APC, more preferablyinto the dendritic cells, can involve co-incubation of the complex ofthe invention with the cells in culture, making use of the intrinsicproperties of the cell penetrating peptide comprised by the complex asdefined herein (i.e.

its internalization ability). Further culture of the cells, e.g. thedendritic cells, thus loaded to induce efficient maturation can includeaddition of cytokines including IL-1β, IL-6, TNFα, PGE2, IFNα, andadjuvants which may include poly-IC, poly-ICLC (i.e. a synthetic complexof carboxymethylcellulose, polyinosinic-polycytidylic acid, andpoly-L-lysine double-stranded RNA), and further TLR agonists and NLR(nucleotide-binding oligomerization domain-like receptors) agonists.

A method for preparing cells, in particular antigen presenting cells,loaded with the complex as defined herein may comprise the steps of:

-   -   (i) transducing or transfecting said cells with the complex of        the invention;    -   (ii) cultivating said cells in a culture medium; and    -   (iii) separating said cells from the culture medium.

Preferably, the cells are loaded with a complex as defined herein,wherein the complex is a polypeptide or a protein and used in theprevention and/or treatment of colorectal cancer.

Preferably, the cells loaded with a complex(es) according as definedherein and used in the prevention and/or treatment of colorectal cancerpresent the at least one antigen or antigenic epitope comprised by saidcomplex at the cell surface in an MHC class I context and/or in an MHCclass II context.

Compositions and Kits According to the Present Invention

According to another aspect, the invention provides a composition foruse in the prevention and/or treatment of colorectal cancer, thecomposition comprising at least one component selected from:

-   -   (i) a complex as described above,    -   (ii) a nucleic acid as described above,    -   (iii) a vector as described above,    -   (iv) a host cell as described above, and    -   (v) a cell loaded with a complex as defined herein as described        above.

Preferably, the composition according to the present invention comprisesthe complex as defined herein.

The composition for use according to the present invention may alsocomprises more than one of the above components (i) to (v). For example,the composition for use according to the present invention may compriseat least two different complexes under (i), at least two differentnucleic acids under (ii), at least two different vectors under (iii), atleast two different host cells under (iv), and/or at least two differentcells under (v); e.g., the composition for use according to theinvention may comprise at least two different complexes (i) and/or atleast two different nucleic acids (ii).

For example, the different complexes (i) comprised by the composition asdescribed above may differ in either component a), i.e. in the cellpenetrating peptides, in component b), i.e. in the antigens or antigenicepitopes or in the subsets of more than one antigen or antigenicepitope, or in component c), i.e. in the TLR peptide agonist or in thesubset of more than one TLR peptide agonist; or the different complexes(i) comprised by the composition as described above may differ in twoout of the three components a), b), and c); or the different complexes(i) comprised by the composition as described above may differ in allthree components a), b), and c) of the complex. Accordingly, thedifferent nucleic acids (ii) comprised by the composition as describedabove may differ in that they encode such different complexes; thedifferent vectors (iii) comprised by the composition as described abovemay differ in that they comprise such different nucleic acids; thedifferent host cells (iv) comprised by the composition as describedabove may differ in that they comprise such different vectors; and thedifferent cells loaded with a complex (v) comprised by the compositionas described above may differ in that they are loaded with suchdifferent complexes.

The present invention also provides a vaccine for use in the preventionand/or treatment of colorectal cancer, the vaccine comprising at leastone component selected from:

-   -   (i) a complex as described above,    -   (ii) a nucleic acid as described above,    -   (iii) a vector as described above,    -   (iv) a host cell as described above, and    -   (v) a cell loaded with a complex as described above.

Preferably, the vaccine for use according to the present inventioncomprises the complex as defined herein.

Thereby, the above details described for the composition for useaccording to the present invention regarding more than one of thecomponents (i) to (v), also apply for the vaccine for use according tothe present invention.

As used in the context of the present invention, the term “vaccine”refers to a biological preparation that provides innate and/or adaptiveimmunity, typically to a particular disease, preferably cancer. Thus, avaccine supports in particular an innate and/or an adaptive immuneresponse of the immune system of a subject to be treated. For example,the antigen or antigenic epitope of the complex as defined hereintypically leads to or supports an adaptive immune response in thepatient to be treated, and the TLR peptide agonist of the complex asdefined herein may lead to or support an innate immune response.

The inventive composition, in particular the inventive vaccine, may alsocomprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicleas defined below for the inventive pharmaceutical composition. In thespecific context of the inventive composition, in particular of theinventive vaccine, the choice of a pharmaceutically acceptable carrieris determined in principle by the manner in which the inventivecomposition, in particular the inventive vaccine, is administered. Theinventive composition, in particular the inventive vaccine, can beadministered, for example, systemically or locally. Routes for systemicadministration in general include, for example, transdermal, oral,parenteral routes, including subcutaneous, intravenous, intramuscular,intraarterial, intradermal and intraperitoneal injections and/orintranasal administration routes. Routes for local administration ingeneral include, for example, topical administration routes but alsointradermal, transdermal, subcutaneous, or intramuscular injections orintralesional, intracranial, intrapulmonal, intracardial, intranodal andsublingual injections. More preferably, inventive composition, inparticular the vaccines, may be administered by an intradermal,subcutaneous, intranodal or intramuscular route. Even more preferably,the inventive composition, in particular the vaccine, may beadministered by subcutaneous, intranodal or intramuscular route.Particularly preferably, the inventive composition, in particular thevaccines, may be administered by subcutaneous or intranodal route. Mostpreferably, the inventive composition, in particular the vaccines may beadministered by subcutaneous route. Inventive composition, in particularthe inventive vaccines, are therefore preferably formulated in liquid(or sometimes in solid) form.

The suitable amount of the inventive composition, in particular theinventive vaccine, to be administered can be determined by routineexperiments with animal models. Such models include, without implyingany limitation, rabbit, sheep, mouse, rat, dog and non-human primatemodels. Preferred unit dose forms for injection include sterilesolutions of water, physiological saline or mixtures thereof. The pH ofsuch solutions should be adjusted to about 7.4. Suitable carriers forinjection include hydrogels, devices for controlled or delayed release,polylactic acid and collagen matrices. Suitable pharmaceuticallyacceptable carriers for topical application include those which aresuitable for use in lotions, creams, gels and the like. If the inventivecomposition, in particular the inventive vaccine, is to be administeredorally, tablets, capsules and the like are the preferred unit dose form.The pharmaceutically acceptable carriers for the preparation of unitdose forms which can be used for oral administration are well known inthe prior art. The choice thereof will depend on secondaryconsiderations such as taste, costs and storability, which are notcritical for the purposes of the present invention, and can be madewithout difficulty by a person skilled in the art.

The inventive composition, in particular the inventive vaccine, canadditionally contain one or more auxiliary substances in order tofurther increase its immunogenicity. A synergistic action of theinventive complex as defined above and of an auxiliary substance, whichmay be optionally contained in the inventive vaccine as described above,is preferably achieved thereby. Depending on the various types ofauxiliary substances, various mechanisms can come into consideration inthis respect. For example, compounds that permit the maturation ofdendritic cells (DCs), for example lipopolysaccharides, TNF-alpha orCD40 ligand, form a first class of suitable auxiliary substances. Ingeneral, it is possible to use as auxiliary substance any agent thatinfluences the immune system in the manner of a “danger signal” (LPS,GP96, etc.) or cytokines, such as GM-CSF, which allow an immune responseproduced by the immune-stimulating adjuvant according to the inventionto be enhanced and/or influenced in a targeted manner. Particularlypreferred auxiliary substances are cytokines, such as monokines,lymphokines, interleukins or chemokines, that further promote the innateimmune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF,G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.

Further additives which may be included in the inventive vaccine areemulsifiers, such as, for example, Tween®; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

The inventive composition, in particular the inventive vaccine, can alsoadditionally contain any further compound, which is known to beimmune-stimulating due to its binding affinity (as ligands) to humanToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, or due to its binding affinity (as ligands) to murineToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to an inventivecomposition, in particular to an inventive vaccine, in this context, maybe CpG nucleic acids, in particular CpG-RNA or CpG-DNA. A CpG-RNA orCpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a double-strandedCpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or adouble-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid is preferablyin the form of CpG-RNA, more preferably in the form of single-strandedCpG-RNA (ss CpG-RNA). The CpG nucleic acid preferably contains at leastone or more (mitogenic) cytosine/guanine dinucleotide sequence(s) (CpGmotif(s)). According to a first preferred alternative, at least one CpGmotif contained in these sequences, in particular the C (cytosine) andthe G (guanine) of the CpG motif, is unmethylated. All further cytosinesor guanines optionally contained in these sequences can be eithermethylated or unmethylated. According to a further preferredalternative, however, the C (cytosine) and the G (guanine) of the CpGmotif can also be present in methylated form.

The present invention also provides a pharmaceutical composition for usein the prevention and/or treatment of colorectal cancer, in particular avaccine composition as described above, and a method for treating asubject, preferably a mammalian subject, and most preferably a humansubject, who is suffering from colorectal cancer.

In particular, the present invention provides a pharmaceuticalcomposition for use in the prevention and/or treatment of colorectalcancer comprising at least one complex as defined herein or at least onecell loaded with a complex as defined herein, and optionally apharmaceutically acceptable carrier and/or vehicle, or any excipient,buffer, stabilizer or other materials well known to those skilled in theart, in particular the pharmaceutical composition comprising at leastone complex as defined herein or at least one cell loaded with a complexas defined herein and a pharmaceutically acceptable carrier.

As a further ingredient, the inventive pharmaceutical composition may inparticular comprise a pharmaceutically acceptable carrier and/orvehicle. In the context of the present invention, a pharmaceuticallyacceptable carrier typically includes the liquid or non-liquid basis ofthe inventive pharmaceutical composition. If the inventivepharmaceutical composition is provided in liquid form, the carrier willtypically be pyrogen-free water; isotonic saline or buffered (aqueous)solutions, e.g phosphate, citrate etc. buffered solutions. Particularlyfor injection of the inventive inventive pharmaceutical composition,water or preferably a buffer, more preferably an aqueous buffer, may beused, containing a sodium salt, preferably at least 30 mM of a sodiumsalt, a calcium salt, preferably at least 0.05 mM of a calcium salt, andoptionally a potassium salt, preferably at least 1 mM of a potassiumsalt. According to a preferred embodiment, the sodium, calcium and,optionally, potassium salts may occur in the form of their halogenides,e.g. chlorides, iodides, or bromides, in the form of their hydroxides,carbonates, hydrogen carbonates, or sulfates, etc. Without being limitedthereto, examples of sodium salts include e.g. NaCl, NaI, NaBr, Na₂CO₃,NaHCO₃, Na₂SO₄, examples of the optional potassium salts include e.g.KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examples of calcium salts includee.g. CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂. Furthermore, organicanions of the aforementioned cations may be contained in the buffer.According to a more preferred embodiment, the buffer suitable forinjection purposes as defined above, may contain salts selected fromsodium chloride (NaCl), calcium chloride (CaCl₂) and optionallypotassium chloride (KCl), wherein further anions may be presentadditional to the chlorides. CaCl₂ can also be replaced by another saltlike KCl. Typically, the salts in the injection buffer are present in aconcentration of at least 30 mM sodium chloride (NaCl), at least 1 mMpotassium chloride (KCl) and at least 0.05 mM calcium chloride (CaCl₂).The injection buffer may be hypertonic, isotonic or hypotonic withreference to the specific reference medium, i.e. the buffer may have ahigher, identical or lower salt content with reference to the specificreference medium, wherein preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cells due toosmosis or other concentration effects. Reference media are e.g. liquidsoccurring in “in vivo” methods, such as blood, lymph, cytosolic liquids,or other body liquids, or e.g. liquids, which may be used as referencemedia in“in vitro” methods, such as common buffers or liquids. Suchcommon buffers or liquids are known to a skilled person. Saline (0.9%NaCl) and Ringer-Lactate solution are particularly preferred as a liquidbasis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to asubject to be treated. The term “compatible” as used herein means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the complex as defined herein as definedabove in such a manner that no interaction occurs which wouldsubstantially reduce the pharmaceutical effectiveness of the inventivepharmaceutical composition under typical use conditions.Pharmaceutically acceptable carriers, fillers and diluents must, ofcourse, have sufficiently high purity and sufficiently low toxicity tomake them suitable for administration to a subject to be treated. Someexamples of compounds which can be used as pharmaceutically acceptablecarriers, fillers or constituents thereof are sugars, such as, forexample, lactose, glucose and sucrose; starches, such as, for example,corn starch or potato starch; cellulose and its derivatives, such as,for example, sodium carboxymethylcellulose, ethylcellulose, celluloseacetate; powdered tragacanth; malt; gelatin; tallow; solid glidants,such as, for example, stearic acid, magnesium stearate; calcium sulfate;vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, intranodaland sublingual injection or infusion techniques. Preferably, theinventive pharmaceutical composition may be administered intradermally,intramuscularly, intranodally or subcutaneously. More preferably theinventive pharmaceutical composition may be administeredintramuscularly, intranodally or subcutaneously. Even more preferablythe inventive pharmaceutical composition may be administeredintranodally or subcutaneously. Most preferably, the inventivepharmaceutical composition may be administered subcutaneously.

Preferably, the inventive pharmaceutical composition may be administeredby parenteral injection, more preferably by subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial, transdermal,intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, intranodal and sublingual injection or via infusiontechniques. Sterile injectable forms of the inventive pharmaceuticalcompositions may be aqueous or oleaginous suspension. These suspensionsmay be formulated according to techniques known in the art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1.3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation of the inventivepharmaceutical composition.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will preferably be in theform of a parenterally acceptable aqueous solution which is pyrogen-freeand has suitable pH, isotonicity and stability. Those of relevant skillin the art are well able to prepare suitable solutions using, forexample, isotonic vehicles such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be included, asrequired. Whether it is a polypeptide, peptide, or nucleic acidmolecule, other pharmaceutically useful compound according to thepresent invention that is to be given to an individual, administrationis preferably in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated.

The inventive pharmaceutical composition as defined above may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient, i.e. the inventivetransporter cargo conjugate molecule as defined above, is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive immunostimulatory composition, particularly its components asdefined above, suspended or dissolved in one or more carriers. Carriersfor topical administration include, but are not limited to, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, theinventive pharmaceutical composition can be formulated in a suitablelotion or cream. In the context of the present invention, suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

In this context, prescription of treatment, e.g. decisions on dosageetc. when using the above pharmaceutical composition is typically withinthe responsibility of general practitioners and other medical doctors,and typically takes account of the disorder to be treated, the conditionof the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in REMINGTON'SPHARMACEUTICAL SCIENCES, 16th edition, Osol, A. (ed), 1980.

Accordingly, the inventive pharmaceutical composition typicallycomprises a “safe and effective amount” of the components of theinventive pharmaceutical composition, in particular of the complex asdefined herein as defined above and/or cells loaded with said complex.As used herein, a “safe and effective amount” means an amount of thecomplex as defined herein that is sufficient to significantly induce apositive modification of a disease or disorder, i.e. an amount of thecomplex as defined herein or cells loaded with said complex, thatelicits the biological or medicinal response in a tissue, system, animalor human that is being sought. An effective amount may be a“therapeutically effective amount” for the alleviation of the symptomsof the disease or condition being treated and/or a “prophylacticallyeffective amount” for prophylaxis of the symptoms of the disease orcondition being prevented. The term also includes the amount of activecomplex sufficient to reduce the progression of the disease, notably toreduce or inhibit the tumor growth or infection and thereby elicit theresponse being sought, in particular such response could be an immuneresponse directed against the antigens or antigenic epitopes comprisedin by the complex (i.e. an “inhibition effective amount”). At the sametime, however, a “safe and effective amount” is small enough to avoidserious side-effects, that is to say to permit a sensible relationshipbetween advantage and risk. The determination of these limits typicallylies within the scope of sensible medical judgment. A “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the complex as defined herein as definedabove, will furthermore vary in connection with the particular conditionto be treated and also with the age and physical condition of thepatient to be treated, the body weight, general health, sex, diet, timeof administration, rate of excretion, drug combination, the activity ofthe specific components a), b), and c) of the complex as defined hereinas defined above, the severity of the condition, the duration of thetreatment, the nature of the accompanying therapy, of the particularpharmaceutically acceptable carrier used, and similar factors, withinthe knowledge and experience of the accompanying doctor. The inventivepharmaceutical composition may be used for human and also for veterinarymedical purposes, preferably for human medical purposes, as apharmaceutical composition in general or as a vaccine.

Pharmaceutical compositions, in particular vaccine compositions, orformulations according to the invention may be administered as apharmaceutical formulation which can contain a complex as defined hereinin any form described herein.

The terms “pharmaceutical formulation” and “pharmaceutical composition”as used in the context of the present invention refer in particular topreparations which are in such a form as to permit biological activityof the active ingredient(s) to be unequivocally effective and whichcontain no additional component which would be toxic to subjects towhich the said formulation would be administered.

In the context of the present invention, an “efficacy” of a treatmentcan be measured based on changes in the course of a disease in responseto a use or a method according to the present invention. For example,the efficacy of a treatment of cancer can be measured by a reduction oftumor volume, and/or an increase of progression free survival time,and/or a decreased risk of relapse post-resection for primary cancer.More specifically for cancer treated by immunotherapy, assessment ofefficacy can be by the spectrum of clinical patterns of antitumorresponse for immunotherapeutic agents through novel immune-relatedresponse criteria (irRC), which are adapted from Response EvaluationCriteria in Solid Tumors (RECIST) and World Health Organization (WHO)criteria (J. Natl. Cancer Inst. 2010, 102(18): 1388-1397). The efficacyof prevention of infectious disease is ultimately assessed byepidemiological studies in human populations, which often correlateswith titres of neutralizing antibodies in sera, and induction ofmultifunctional pathogen specific T cell responses. Preclinicalassessment can include resistance to infection after challenge withinfectious pathogen. Treatment of an infectious disease can be measuredby inhibition of the pathogen's growth or elimination of the pathogen(and, thus, absence of detection of the pathogen), correlating withpathogen specific antibodies and/or T cell immune responses.

Pharmaceutical compositions, in particular vaccine compositions, orformulations according to the invention may also be administered as apharmaceutical formulation which can contain antigen presenting cellsloaded with a complex according to the invention in any form describedherein.

The vaccine and/or the composition for use according to the presentinvention may also be formulated as pharmaceutical compositions and unitdosages thereof, in particular together with a conventionally employedadjuvant, immunomodulatory material, carrier, diluent or excipient asdescribed above and below, and in such form may be employed as solids,such as tablets or filled capsules, or liquids such as solutions,suspensions, emulsions, elixirs, or capsules filled with the same, allfor oral use, or in the form of sterile injectable solutions forparenteral (including subcutaneous and intradermal) use by injection orcontinuous infusion.

In the context of the present invention, in particular in the context ofa pharmaceutical composition and vaccines according to the presentinvention, injectable compositions are typically based upon injectablesterile saline or phosphate-buffered saline or other injectable carriersknown in the art. Such pharmaceutical compositions and unit dosage formsthereof may comprise ingredients in conventional proportions, with orwithout additional active compounds or principles, and such unit dosageforms may contain any suitable effective amount of the active ingredientcommensurate with the intended daily dosage range to be employed.

Examples of suitable adjuvants and/or immunomodulatory materials in thecontext of the present invention include MPL® (Corixa), aluminum-basedminerals including aluminum compounds (generically called Alum), ASO1-4,MF59, CalciumPhosphate, Liposomes, Iscom, polyinosinic:polycytidylicacid (polyIC), including its stabilized form poly-ICLC (Hiltonol), CpGoligodeoxynucleotides, Granulocyte-macrophage colony-stimulating factor(GM-CSF), lipopolysaccharide (LPS), Montanide, polylactide co-glycolide(PLG), Flagellin, Soap Bark tree saponins (Q521), amino alkylglucosamide compounds (e.g. RC529), two component antibacterial peptideswith synthetic oligodeoxynucleotides (e.g. IC31), Imiquimod, Resiquimod,Immunostimulatory sequences (ISS), monophosphoryl lipid A (MPLA),Fibroblast-stimulating lipopeptide (FSL1), and anti-CD40 antibodies.

Compositions, in particular pharmaceutical compositions and vaccines,for use according to the present invention may be liquid formulationsincluding, but not limited to, aqueous or oily suspensions, solutions,emulsions, syrups, and elixirs. The compositions may also be formulatedas a dry product for reconstitution with water or other suitable vehiclebefore use. Such liquid preparations may contain additives including,but not limited to, suspending agents, emulsifying agents, non-aqueousvehicles and preservatives. Suspending agents include, but are notlimited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup,gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminumstearate gel, and hydrogenated edible fats. Emulsifying agents include,but are not limited to, lecithin, sorbitan monooleate, and acacia.Preservatives include, but are not limited to, methyl or propylp-hydroxybenzoate and sorbic acid. Dispersing or wetting agents includebut are not limited to poly(ethylene glycol), glycerol, bovine serumalbumin, Tween®, Span®.

Compositions, in particular pharmaceutical compositions and vaccines,for use according to the present invention may also be formulated as adepot preparation, which may be administered by implantation or byintramuscular injection.

Compositions, in particular pharmaceutical compositions and vaccines,for use according to the present invention may also be solidcompositions, which may be in the form of tablets or lozenges formulatedin a conventional manner. For example, tablets and capsules for oraladministration may contain conventional excipients including, but notlimited to, binding agents, fillers, lubricants, disintegrants andwetting agents. Binding agents include, but are not limited to, syrup,accacia, gelatin, sorbitol, tragacanth, mucilage of starch andpolyvinylpyrrolidone. Fillers include, but are not limited to, lactose,sugar, microcrystalline cellulose, maizestarch, calcium phosphate, andsorbitol. Lubricants include, but are not limited to, magnesiumstearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants include, but are not limited to, potato starch and sodiumstarch glycollate. Wetting agents include, but are not limited to,sodium lauryl sulfate. Tablets may be coated according to methods wellknown in the art.

Compositions, in particular pharmaceutical compositions and vaccines,for use according to the present invention may also be administered insustained release forms or from sustained release drug delivery systems.

Moreover, the compositions, in particular pharmaceutical compositionsand vaccines, for use according to the present invention may be adaptedfor delivery by repeated administration.

Further materials as well as formulation processing techniques and thelike, which are useful in the context of compositions, in particularpharmaceutical compositions and vaccines, for use according to thepresent invention or in the context of their preparation are set out in“Part 5 of Remington's “The Science and Practice of Pharmacy”, 22ndEdition, 2012, University of the Sciences in Philadelphia, LippincottWilliams & Wilkins”.

In a further aspect, the present invention also relates to akit-of-parts for use in the prevention and/or treatment of colorectalcancer, the kit of parts comprising at least one of:

-   -   (i) a complex as described above,    -   (ii) a nucleic acid as described above,    -   (iii) a vector as described above,    -   (iv) a host cell as described above, and    -   (v) a cell loaded with a complex as described above.

In particular, the kit-of-parts of the invention may comprise more thanone component (i) to (v). For example, the kit-of-parts according to thepresent invention may comprise at least two different complexes under(i), at least two different nucleic acids under (ii), at least twodifferent vectors under (iii), at least two different host cells under(iv), and/or at least two different cells under (v); e.g., thekit-of-parts of the invention may comprise at least two differentcomplexes (i) and/or at least two different nucleic acids (ii).

For example, the different complexes (i) comprised by the kit-of-partsas described above may differ in either component a), i.e. in the cellpenetrating peptides, in component b), i.e. in the antigens or antigenicepitopes or in the subsets of more than one antigen or antigenicepitope, or in component c), i.e. in the TLR peptide agonist or in thesubset of more than one TLR peptide agonist; or the different complexes(i) comprised by the kit-of-parts as described above may differ in twoout of the three components a), b), and c); or the different complexes(i) comprised by the kit-of-parts as described above may differ in allthree components a), b), and c) of the complex. Accordingly, thedifferent nucleic acids (ii) comprised by the kit-of-parts as describedabove may differ in that they encode such different complexes; thedifferent vectors (iii) comprised by the kit-of-parts as described abovemay differ in that they comprise such different nucleic acids; thedifferent host cells (iv) comprised by the kit-of-parts as describedabove may differ in that they comprise such different vectors; and thedifferent cells loaded with a complex (v) comprised by the kit-of-partsas described above may differ in that they are loaded with suchdifferent complexes.

The various components of the kit-of-parts may be packaged in one ormore containers. The above components may be provided in a lyophilizedor dry form or dissolved in a suitable buffer. The kit may also compriseadditional reagents including, for instance, preservatives, growthmedia, and/or buffers for storage and/or reconstitution of theabove-referenced components, washing solutions, and the like. Inaddition, the kit-of-parts according to the present invention mayoptionally contain instructions of use.

Moreover, the present invention also provides a vaccination kit fortreating, preventing and/or stabilizing colorectal cancer, comprisingthe pharmaceutical composition as described herein or a vaccine asdescribed herein and instructions for use of said pharmaceuticalcomposition or of said vaccine in the prevention and/or treatment ofcolorectal cancer.

Thus, the present invention also provides a kit comprising the complexas described herein, the cell as described herein, the composition asdescribed herein, the vaccine as described herein, and/or thepharmaceutical composition as described herein.

Preferably, such a kit further comprises a package insert or instructionleaflet with directions to treat colorectal cancer by using the complexfor use according to the present invention as described herein, the cellas described herein, the composition as described herein, the vaccine asdescribed herein, and/or the pharmaceutical composition as describedherein.

Use and Methods According to the Invention

In another aspect, the present invention provides the use of any one of:(i) a complex as described herein, and/or (ii) cells, such asantigen-presenting cells, loaded with a complex as described herein,(for the preparation of a medicament) for the prevention, treatment orstabilization of colorectal cancer. Accordingly, the present inventionprovides any one of: (i) a complex as described herein, and/or (ii)cells, such as antigen-presenting cells, loaded with a complex asdescribed herein, for use in the prevention, treatment or stabilizationof colorectal cancer.

The present invention also provides a complex for use according to thepresent invention, which allows the transport and presentation of the atleast one antigen or antigenic epitope comprised by the complex at thecell surface of antigen presenting cells in an MHC class I and/or MHCclass II context, for use in vaccination and/or immunotherapy.

According to another aspect, the present invention provides a method ofpreventing, treating or repressing colorectal cancer, wherein saidmethod comprises administering any one of: (i) a complex of theinvention, (ii) cells, such as antigen-presenting cells, loaded with acomplex of the invention, or (iii) a pharmaceutical formulation of (i)to (ii), to said subject.

Moreover, the present invention provides a method for eliciting orimproving, in a subject, an immune response against one or multipleepitopes that is dependent on CD4+ helper T cells and/or CD8+ cytotoxicT cells, wherein said method comprises administering any one of: (i) acomplex for use according to the present invention, and/or (ii) cells,such as antigen-presenting cells, loaded with said complex, or (iii) apharmaceutical formulation of (i) to (ii), to said subject.

An immune response that is dependent on CD4+ and/or CD8+ response can bedetermined by evaluating an inflammatory response, a pro-inflammatorycytokine response, including an increase in the expression of one ormore of IFN-γ, TNF-α and IL-2 mRNA or protein relative to the levelbefore administration of the compounds of the invention. It can also bemeasured by an increase in the frequency or absolute number ofantigen-specific T cells after administration of the compounds of theinvention, measured by HLA-peptide multimer staining, ELISPOT assays,and delayed type hypersensitivity tests. It can also be indirectlymeasured by an increase in antigen-specific serum antibodies that aredependent on antigen-specific T helper cells.

The present invention also provides a method for eliciting or improving,in a subject, an immune response against one or multiple antigens orantigenic epitopes that is restricted by multiple MHC class I moleculesand/or multiple MHC class II molecules, wherein said method comprisesadministering any one of: (i) a complex for use according to the presentinvention, and/or (ii) cells, such as antigen-presenting cells, loadedwith said complex, or (iii) a pharmaceutical formulation of (i) to (ii),to said subject.

A method for eliciting or improving, in a subject, an immune responseagainst multiple epitopes as described herein, that is restricted bymultiple MHC class I molecules and/or multiple MHC class II moleculescan be determined by evaluating a cytokine response, including anincrease in the expression of one or more of IFN-γ, TNF-α and IL-2 mRNAor protein relative to the level before administration of the compoundsof the invention, after in vitro stimulation of T cells with individualpeptides binding to discrete MHC class I and class II molecules onantigen presenting cells. Restriction to different MHC molecules canalso be validated by using antigen presenting cells expressing differentMHC molecules, or by using MHC blocking antibodies. It can also bemeasured by an increase in the frequency or absolute number ofantigen-specific T cells after administration of the compounds of theinvention, measured by HLA-peptide multimer staining, which usesmultimers assembled with discrete MHC molecules.

Preferably, in the methods for eliciting or improving an immune responseagainst one or multiple antigens or antigenic epitopes according to thepresent invention, the immune response is directed against one ormultiple epitopes of a tumor-associated antigen or a tumor-specificantigen as, for instance, a combination of epitopes as described herein.

Alternatively or additionally, the immune response may be directedagainst multiple epitopes of an antigenic protein from a pathogen.

The methods according to the present invention as described herein, maybe for eliciting or improving, in a subject, an immune response againstone or multiple epitopes that is restricted by MHC class I moleculesand/or MHC class II molecules.

In particular, the present invention thus provides a method forpreventing and/or treating colorectal cancer or initiating, enhancing orprolonging an anti-tumor-response in a subject in need thereofcomprising administering to the subject a complex comprising:

-   -   a cell penetrating peptide;    -   at least one antigen or antigenic epitope; and    -   at least one TLR peptide agonist,    -   wherein the components a)-c) are covalently linked.

In such a method it is preferred that the complex for use according tothe present invention as described herein, the cell as described herein,the composition as described herein, the vaccine as described herein,and/or the pharmaceutical composition as described herein isadministered to the subject.

Preferably, the subject has colorectal cancer and/or was diagnosed withcolorectal cancer. In another aspect, the present invention provides theuse of any one of: (i) a complex as described herein, and/or (ii) cells,such as antigen-presenting cells, loaded with the complex as describedherein, for the preparation of an imaging composition for imagingtechniques in the context of (diagnosis of) colorectal cancer or for thepreparation of a diagnosis composition (“diagnostic compositions”) fordiagnosing colorectal cancer. A diagnostic composition for diagnosingcolorectal cancer according to the present invention comprises at leastone component selected from:

-   -   (i) a complex as described above,    -   (ii) a nucleic acid as described above,    -   (iii) a vector as described above,    -   (iv) a host cell as described above, and    -   (v) a cell loaded with a complex as described above.

Preferably, the diagnostic composition according to the presentinvention comprises the complex as described above.

In particular, the complex for use according to the present invention,the cell, such as antigen-presenting cell, loaded with the complex foruse according to the present invention, the inventive composition, theinventive pharmaceutical composition or the inventive vaccine or, mostpreferably, the inventive diagnostic composition may be utilized indiagnosis as a diagnostic tool, e.g. in (in vivo or in vitro) assays,e.g. in immunoassays, to detect, prognose, diagnose, or monitorcolorectal cancer.

As an example, (in vitro) assays may be performed by delivering thecomplex for use according to the present invention, the cell, such asantigen-presenting cell, loaded with the complex for use according tothe present invention, the inventive composition, the inventivepharmaceutical composition or the inventive vaccine or, most preferably,the inventive diagnostic composition to target cells typically selectedfrom e.g. cultured animal cells, human cells or micro-organisms, and tomonitor the cell response by biophysical methods typically known to askilled person. The target cells typically used therein may be culturedcells (in vitro), e.g. cells isolated from human or animal body, such asblood cells isolated from human or animal body, or in vivo cells, i.e.cells composing the organs or tissues of living animals or humans, ormicroorganisms found in living animals or humans. Particularlypreferable in this context are so called markers or labels, which may becontained in the complex for use according to the present invention and,in particular, in the diagnostic composition according to the presentinvention.

According to a further aspect, the invention provides a method ofdiagnosing colorectal cancer in a subject, wherein said method comprisesadministering any one of: (i) a complex of the invention, (ii) cells,such as antigen-presenting cells, loaded with the complex of theinvention, or (iii) a pharmaceutical formulation of (i) to (ii), to saidsubject or to said subject's sample ex vivo.

Preferably, uses and methods according to the present invention compriseadministration of a complex for use according to the invention.

Moreover, uses and methods according to the present invention compriseadministration of more than one complex, cells, or pharmaceuticalformulation according to the invention. For example, in the uses andmethods according to the present invention, at least two differentcomplexes are used or administered, wherein each complex comprises atleast one antigen or antigenic epitope and said antigen or antigenicepitope or (if more than one antigen or antigenic epitope is comprisedby said complex) said subset of antigens or antigenic epitopes aredifferent between the two complexes.

For example, the different complexes (i) comprised by the composition asdescribed above may differ in either component a), i.e. in the cellpenetrating peptides, in component b), i.e. in the antigens or antigenicepitopes or in the subsets of more than one antigen or antigenicepitope, or in component c), i.e. in the TLR peptide agonist or in thesubset of more than one TLR peptide agonist; or the different complexes(i) comprised by the composition as described above may differ in twoout of the three components a), b), and c); or the different complexes(i) comprised by the composition as described above may differ in allthree components a), b), and c) of the complex. Accordingly, thedifferent nucleic acids (ii) comprised by the composition as describedabove may differ in that they encode such different complexes; thedifferent vectors (iii) comprised by the composition as described abovemay differ in that they comprise such different nucleic acids; thedifferent host cells (iv) comprised by the composition as describedabove may differ in that they comprise such different vectors; and thedifferent cells loaded with a complex (v) comprised by the compositionas described above may differ in that they are loaded with suchdifferent complexes.

Moreover, in the uses and methods according to the present invention,the cells according to the present invention may be antigen presentingcells, in particular dendritic cells, more preferably dendritic cellsfrom the subject to be treated.

Mode of Administration

The complex for use according to the present invention; the cell, suchas antigen-presenting cell, loaded with the complex for use according tothe present invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may be administeredin any manner as described above, including enterally, such as orally orrectally, and parenterally, such as intravenously or combinationsthereof. Parenteral administration includes, but is not limited to,intravenous, intra-arterial, intra-peritoneal, subcutaneous, intradermaland intramuscular. Preferably, the complex for use according to thepresent invention; the cell, such as antigen-presenting cell, loadedwith the complex for use according to the present invention; theinventive composition; the inventive pharmaceutical composition and/orthe inventive vaccine are administered via an enteral route ofadministration, such as oral, sublingual and rectal. The complex for useaccording to the present invention; the cell, such as antigen-presentingcell, loaded with the complex for use according to the presentinvention; the inventive composition; the inventive pharmaceuticalcomposition or the inventive vaccine may also be preferably administeredvia topical, intratumoral, intradermal, subcutaneous, intramuscular,intranasal, or intranodal route. The complex for use according to thepresent invention; the cell, such as antigen-presenting cell, loadedwith the complex for use according to the present invention; theinventive composition; the inventive pharmaceutical composition or theinventive vaccine may also be administered in the form of an implant,which allows slow release of the compositions as well as a slowcontrolled i.v. infusion. For example, the complex for use according tothe present invention; the cell, such as antigen-presenting cell, loadedwith the complex for use according to the present invention; theinventive composition; the inventive pharmaceutical composition or theinventive vaccine may be administered subcutaneously.

The administration of complex for use according to the presentinvention; the cell, such as antigen-presenting cell, loaded with thecomplex for use according to the present invention; the inventivecomposition; the inventive pharmaceutical composition or the inventivevaccine may require multiple successive injections/administrations.Thus, the administration may be repeated at least two times, for exampleonce as primary immunization injections/administration and, later, asbooster injections/administration.

In particular, the complex for use according to the present invention;the cell, such as antigen-presenting cell, loaded with the complex foruse according to the present invention; the inventive composition; theinventive pharmaceutical composition or the inventive vaccine may beadministered repeatedly or continuously. The complex for use accordingto the present invention; the cell, such as antigen-presenting cell,loaded with the complex for use according to the present invention; theinventive composition; the inventive pharmaceutical composition or theinventive vaccine may be administered repeatedly or continuously for aperiod of at least 1, 2, 3, or 4 weeks; 2, 3, 4, 5, 6, 8, 10, or 12months; or 2, 3, 4, or 5 years.

Moreover, the cell penetrating peptide, components a), b), and c), i.e.the at least one antigen or antigenic epitope and the at least one TLRpeptide agonist, composing the complex for use according to the presentinvention may be contained in separate compositions which are mixed justbefore administration or which are administered simultaneously to thesubject in need thereof.

According to one approach, the complex for use according to the presentinvention; the cell, such as antigen-presenting cell, loaded with thecomplex for use according to the present invention; the inventivecomposition; the inventive pharmaceutical composition or the inventivevaccine may be administered directly to a patient using theadministration routes as described above, in particular forpharmaceutical compositions. Alternatively, the complex for useaccording to the present invention; the cell, such as antigen-presentingcell, loaded with the complex for use according to the presentinvention; the inventive composition; the inventive pharmaceuticalcomposition or the inventive vaccine may be administered to a patientusing an ex vivo approach, e.g. by introducing the pharmaceuticalcomposition, the vaccine or the inventive transporter cargo conjugatemolecule as defined above into cells, preferably autologous cells, i.e.cells derived from the patient to be treated, and transplanting thesecells into the site of the patient to be treated, optionally subsequentto storing and/or culturing these cells prior to treatment.

The dosage administered, as single or multiple doses, to an individualwill vary depending upon a variety of factors, including pharmacokineticproperties, subject conditions and characteristics (sex, age, bodyweight, health, size), extent of symptoms, concurrent treatments,frequency of treatment and the effect desired.

Typically, for cancer treatment, the therapeutically effective dose of acomplex for use according to the present invention is from about 0.01 mgto 5 mg per injection, in particular from about 0.1 mg to 2 mg perinjection, or from about 0.01 nmol to 1 mmol per injection, inparticular from 1 nmol to 1 mmol per injection, preferably from 1 □molto 1 mmol per injection.

Typically, for cancer treatment, the therapeutically effective dose ofan antigen presenting cell loaded with a complex for use according tothe present invention is from about 0.2 million cells to 2 million cellsper injection.

Combination Therapy

The administration of the complex for use according to the presentinvention; the cell, such as antigen-presenting cell, loaded with thecomplex for use according to the present invention; the inventivecomposition; the inventive pharmaceutical composition or the inventivevaccine in the methods and uses according to the invention can becarried out alone or in combination with a co-agent useful for treatingand/or stabilizing colorectal cancer.

For instance, in the case of treatment, prevention, or stabilization ofa colorectal cancer, the administration of the pharmaceuticalcompositions in the methods and uses according to the invention can becarried out in combination with substances used in conventionalchemotherapy directed against solid colorectal tumors and for control ofestablishment of metastases or any other molecule that act by triggeringprogrammed cell death e.g. for example a co-agent selected from TumorNecrosis Family Members including, but not limited, to Fas Ligand andtumor necrosis factor (TNF)-related apoptosis inducing (TRAIL) ligand.According to a further embodiment, the administration of the complex foruse according to the present invention; the cell, such asantigen-presenting cell, loaded with the complex for use according tothe present invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine in the methods anduses according to the present invention can be carried out in parallelof radiotherapy.

The invention encompasses the administration of the complex for useaccording to the present invention; the cell, such as antigen-presentingcell, loaded with the complex for use according to the presentinvention; the inventive composition; the inventive pharmaceuticalcomposition or the inventive vaccine, wherein it is administered to asubject prior to, simultaneously or sequentially with other therapeuticregimens or co-agents useful for treating, and/or stabilizing acolorectal cancer and/or preventing colorectal cancer relapsing (e.g.multiple drug regimens), in a therapeutically effective amount. Saidcomplex, cell, composition, vaccine or pharmaceutical composition, thatis administered simultaneously with said co-agents can be administeredin the same or different composition(s) and by the same or differentroute(s) of administration.

Said other therapeutic regimens or co-agents may be selected from thegroup consisting of radiation therapy, chemotherapy, surgery, targetedtherapy (including small molecules, peptides and monoclonal antibodies),and anti-angiogenic therapy. Anti-angiogenic therapy is defined hereinas the administration of an agent that directly or indirectly targetstumor-associated vasculature.

Accordingly, the present invention also provides a combination of

-   -   (i) a complex as defined herein; and    -   (ii) a chemotherapeutic agent, a targeted drug and/or an        immunotherapeutic agent, such as an immune checkpoint modulator,

for use in the prevention and/or treatment of colorectal cancer.

Traditional chemotherapeutic agents are cytotoxic, i.e. they act bykilling cells that divide rapidly, one of the main properties of mostcancer cells. Preferred chemotherapeutic agents for combination with thecomplex as defined herein are such chemotherapeutic agents known to theskilled person for treatment of colorectal cancer. Preferredchemotherapeutic agents for combination include 5-Fluorouracil (5-FU),Capecitabine (Xeloda®), Irinotecan (Camptosar®) and Oxaliplatin(Eloxatin®). It is also preferred that the complex as defined herein iscombined with a combined chemotherapy, preferably selected from (i)FOLFOX (5-FU, leucovorin, and oxaliplatin); (ii) CapeOx (Capecitabineand oxaliplatin); (iii) 5-FU and leucovorin; (iv) FOLFOXIRI (leucovorin,5-FU, oxaliplatin, and irinotecan); and (v) FOLFIRI (5-FU, leucovorin,and irinotecan). In non-spread cancer, a combination with (i) FOLFOX(5-FU, leucovorin, and oxaliplatin); (ii) CapeOx (Capecitabine andoxaliplatin); or (iii) 5-FU and leucovorinis preferred. For cancer thathas spread, a combination with (iv) FOLFOXIRI (leucovorin, 5-FU,oxaliplatin, and irinotecan); (i) FOLFOX (5-FU, leucovorin, andoxaliplatin); or (v) FOLFIRI (5-FU, leucovorin, and irinotecan) ispreferred.

Targeted drugs for combination with the complex as defined herein fortreatment of colorectal cancer include VEGF-targeted drugs andEGFR-targeted drugs. Preferred examples of VEGF-targeted drugs includeBevacizumab (Avastin®), ramucirumab (Cyramza®) or ziv-aflibercept(Zaltrap®). Preferred examples of EGFR-targeted drugs include Cetuximab(Erbitux®), panitumumab (Vectibix®) or Regorafenib (Stivarga®).

Immunotherapeutic agents for combination with the complex as definedherein for treatment of colorectal cancer include vaccines, chimericantigen receptors (CARs), checkpoint modulators and oncolytic virustherapies.

Preferred vaccines for combination with the complex as defined hereinfor treatment of colorectal cancer include TroVax, OncoVax, IMA910,ETBX-011, MicOryx, EP-2101, MKC1106-PP, CDX-1307, V934/V935,MelCancerVac, Imprime PGG, FANG, Tecemotide, AlloStim, DCVax, GI-6301,AVX701, OCV-C02.

Artificial T cell receptors (also known as chimeric T cell receptors,chimeric immunoreceptors, chimeric antigen receptors (CARs)) areengineered receptors, which graft an arbitrary specificity onto animmune effector cell. Artificial T cell receptors (CARs) are preferredin the context of adoptive cell transfer. To this end, T cells areremoved from a patient and modified so that they express receptorsspecific to colorectal cancer. The T cells, which can then recognize andkill the cancer cells, are reintroduced into the patient.

As used herein, the term “immune checkpoint modulator” (also referred toas “checkpoint modulator”) refers to a molecule or to a compound thatmodulates (e.g., totally or partially reduces, inhibits, interfereswith, activates, stimulates, increases, reinforces or supports) thefunction of one or more checkpoint molecules. Thus, an immune checkpointmodulator may be an “immune checkpoint inhibitor” (also referred to as“checkpoint inhibitor” or “inhibitor”) or an “immune checkpointactivator” (also referred to as “checkpoint activator” or “activator”).An “immune checkpoint inhibitor” (also referred to as “checkpointinhibitor” or “inhibitor”) totally or partially reduces, inhibits,interferes with, or negatively modulates the function of one or morecheckpoint molecules. An “immune checkpoint activator” (also referred toas “checkpoint activator” or “activator”) totally or partiallyactivates, stimulates, increases, reinforces, supports or positivelymodulates the function of one or more checkpoint molecules. Immunecheckpoint modulators are typically able to modulate (i) self-toleranceand/or (ii) the amplitude and/or the duration of the immune response.Preferably, the immune checkpoint modulator used according to thepresent invention modulates the function of one or more human checkpointmolecules and is, thus, a “human checkpoint inhibitor”.

Checkpoint molecules are molecules, such as proteins, are typicallyinvolved in immune pathways and, for example, regulate T-cellactivation, T-cell proliferation and/or T-cell function. Accordingly,the function of checkpoint molecules, which is modulated (e.g., totallyor partially reduced, inhibited, interfered with, activated, stimulated,increased, reinforced or supported) by checkpoint modulators, istypically the (regulation of) T-cell activation, T-cell proliferationand/or T cell function. Immune checkpoint molecules thus regulate andmaintain self-tolerance and the duration and amplitude of physiologicalimmune responses. Many of the immune checkpoint molecules belong to theB7:CD28 family or to the tumor necrosis factor receptor (TNFR) superfamily and, by the binding of specific ligands, activate signalingmolecules that are recruited to the cytoplasmic domain(cf. Susumu Suzukiet al., 2016: Current status of immunotherapy. Japanese Journal ofClinical Oncology, 2016: doi: 10.1093/jjco/hyv201 [Epub ahead of print];in particular Table 1).

Preferably, the immune checkpoint modulator for combination with thecomplex as defined herein for treatment of colorectal cancer is anactivator or an inhibitor of one or more immune checkpoint pointmolecule(s) selected from CD27, CD28, CD40, CD122, CD137, OX40, GITR,ICOS, A2AR, B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1,TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, GITR, TNFRand/or FasR/DcR3; or an activator or an inhibitor of one or more ligandsthereof.

More preferably, the immune checkpoint modulator is an activator of a(co-)stimulatory checkpoint molecule or an inhibitor of an inhibitorycheckpoint molecule or a combination thereof. Accordingly, the immunecheckpoint modulator is more preferably (i) an activator of CD27, CD28,CD40, CD122, CD137, OX40, GITR and/or ICOS or (ii) an inhibitor of A2AR,B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2,TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/orFasR/DcR3.

Even more preferably, the immune checkpoint modulator is an inhibitor ofan inhibitory checkpoint molecule (but preferably no inhibitor of astimulatory checkpoint molecule). Accordingly, the immune checkpointmodulator is even more preferably an inhibitor of A2AR, B7-H3, B7-H4,BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2, TIM-3, VISTA, CEACAM1,GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or DcR3 or of a ligandthereof.

It is also preferred that the immune checkpoint modulator is anactivator of a stimulatory or costimulatory checkpoint molecule (butpreferably no activator of an inhibitory checkpoint molecule).Accordingly, the immune checkpoint modulator is more preferably anactivator of CD27, CD2S, CD40, CD122, CD137, OX40, GITR and/or ICOS orof a ligand thereof.

It is even more preferred that the immune checkpoint modulator is amodulator of the CD40 pathway, of the IDO pathway, of the CTLA-4 pathwayand/or of the PD-1 pathway. In particular, the immune checkpointmodulator is preferably a modulator of CD40, CTLA-4, PD-L1, PD-L2, PD-1and/or IDO, more preferably the immune checkpoint modulator is aninhibitor of CTLA-4, PD-L1, PD-L2, PD-1 and/or IDO or an activator ofCD40, even more preferably the immune checkpoint modulator is aninhibitor of CTLA-4, PD-L1, PD-1 and/or IDO and most preferably theimmune checkpoint modulator is an inhibitor of CTLA-4 and/or PD-1.

Accordingly, the checkpoint modulator for combination with the complexas defined herein for treatment of colorectal cancer may be selectedfrom known modulators of the CD40 pathway, the CTLA-4 pathway or thePD-1 pathway. Preferred inhibitors of the CTLA-4 pathway and of the PD-1pathway include the monoclonal antibodies Yervoy® (Ipilimumab; BristolMyers Squibb) and Tremelimumab (Pfizer/Medlmmune) as well as Opdivo®(Nivolumab; Bristol Myers Squibb), Keytruda® (Pembrolizumab; Merck),Durvalumab (Medlmmune/AstraZeneca), MEDI4736 (AstraZeneca; cf. WO2011/066389 A1), MPDL3280A (Roche/Genentech; cf. U.S. Pat. No. 8,217,149B2), Pidilizumab (CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca),MSB-0010718C (Merck), MIH1 (Affymetrix) and Lambrolizumab (e.g.disclosed as hPD109A and its humanized derivatives h409All, h409A16 andh409A17 in WO2008/156712; Hamid et al., 2013; N. Engl. J. Med. 369:134-144). More preferred checkpoint inhibitors include the CTLA-4inhibitors Yervoy® (Ipilimumab; Bristol Myers Squibb) and Tremelimumab(Pfizer/Medlmmune) as well as the PD-inhibitors Opdivo® (Nivolumab;Bristol Myers Squibb), Keytruda® (Pembrolizumab; Merck), Pidilizumab(CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca), AMP-224 andLambrolizumab (e.g. disclosed as hPD109A and its humanized derivativesh409All, h409A16 and h409A17 in WO2008/156712; Hamid O. et al., 2013; N.Engl. J. Med. 369: 134-144).

It is also preferred that the immune checkpoint modulator forcombination with the complex as defined herein for treatment ofcolorectal cancer is selected from the group consisting ofPembrolizumab, Ipilimumab, Nivolumab, MPDL3280A, MEDI4736, Tremelimumab,Avelumab, PDR001, LAG525, INCB24360, Varlilumab, Urelumab, AMP-224 andCM-24.

Oncolytic viruses are engineered to cause cell lysis by replicating intumors, thus activating an antitumor immune response. An oncolytic virustherapy for combination with the complex as defined herein for treatmentof colorectal cancer is preferably selected from the group consisting ofJX594 (Thymidine Kinase-Deactivated Vaccinia Virus), ColoAd1(adenovirus), NV1020 (HSV-derived), ADXS11-001 (attenuated Listeriavaccine), Reolysin® (special formulation of the human reovirus), PANVAC(recombinant vaccinia-virus CEA-MUC-1-TRICOM), Ad5-hGCC-PADRE(recombinant adenovirus vaccine) and vvDD-CDSR (vaccinia virus).

Preferably, (i) the complex and (ii) the chemotherapeutic agent, thetargeted drug and/or the immunotherapeutic agent, such as an immunecheckpoint modulator, are administered at about the same time.

“At about the same time”, as used herein, means in particularsimultaneous administration or that directly after administration of (i)the chemotherapeutic agent, the targeted drug and/or theimmunotherapeutic agent, such as an immune checkpoint modulator, (ii)the complex is administered or directly after administration of (i) thecomplex (ii) the chemotherapeutic agent, the targeted drug and/or theimmunotherapeutic agent, such as an immune checkpoint modulator, isadministered. The skilled person understands that “directly after”includes the time necessary to prepare the second administration—inparticular the time necessary for exposing and disinfecting the locationfor the second administration as well as appropriate preparation of the“administration device” (e.g., syringe, pump, etc.). Simultaneousadministration also includes if the periods of administration of (i) thecomplex and of (ii) the chemotherapeutic agent, the targeted drug and/orthe immunotherapeutic agent, such as an immune checkpoint modulator,overlap or if, for example, one component is administered over a longerperiod of time, such as 30 min, 1 h, 2 h or even more, e.g. by infusion,and the other component is administered at some time during such a longperiod. Administration of (i) the complex and of (ii) thechemotherapeutic agent, the targeted drug and/or the immunotherapeuticagent, such as an immune checkpoint modulator, at about the same time isin particular preferred if different routes of administration and/ordifferent administration sites are used.

It is also preferred that (i) the complex and (ii) the chemotherapeuticagent, the targeted drug and/or the immunotherapeutic agent, such as animmune checkpoint modulator, are administered consecutively. This meansthat (i) the complex is administered before or after (ii) thechemotherapeutic agent, the targeted drug and/or the immunotherapeuticagent, such as an immune checkpoint modulator. In consecutiveadministration, the time between administration of the first componentand administration of the second component is preferably no more thanone week, more preferably no more than 3 days, even more preferably nomore than 2 days and most preferably no more than 24 h. It isparticularly preferred that (i) the complex and (ii) thechemotherapeutic agent, the targeted drug and/or the immunotherapeuticagent, such as an immune checkpoint modulator, are administered at thesame day with the time between administration of the first component(the checkpoint modulator of the complex) and administration of thesecond component (the other of the checkpoint modulator and the complex)being preferably no more than 6 hours, more preferably no more than 3hours, even more preferably no more than 2 hours and most preferably nomore than 1 h.

Preferably, (i) the complex and (ii) the chemotherapeutic agent, thetargeted drug and/or the immunotherapeutic agent, such as an immunecheckpoint modulator, are administered via the same route ofadministration. It is also preferred that (i) the complex and (ii) thechemotherapeutic agent, the targeted drug and/or the immunotherapeuticagent, such as an immune checkpoint modulator, are administered viadistinct routes of administration.

Moreover, (i) the complex and (ii) the chemotherapeutic agent, thetargeted drug and/or the immunotherapeutic agent, such as an immunecheckpoint modulator, are preferably provided in distinct compositions.Alternatively, (i) the complex and (ii) the chemotherapeutic agent, thetargeted drug and/or the immunotherapeutic agent, such as an immunecheckpoint modulator, are preferably provided in the same composition.

Accordingly, the present invention provides a pharmaceutical formulationcomprising a complex for use according to the invention or a cell foruse according to the invention, in particular an antigen-presenting cellfor use according to the invention, combined with at least one co-agentuseful for treating and/or stabilizing a cancer and/or preventingcolorectal cancer relapsing, and at least one pharmaceuticallyacceptable carrier.

Moreover, the complex for use according to the present invention; thecell, such as antigen-presenting cell, loaded with the complex for useaccording to the present invention; the inventive composition; theinventive pharmaceutical composition or the inventive vaccine can beadministered after surgery where solid tumors have been removed as aprophylaxis against relapsing and/or metastases.

Moreover, the administration of the imaging or diagnosis composition inthe methods and uses according to the invention can be carried out aloneor in combination with a co-agent useful for imaging and/or diagnosingcolorectal cancer.

Subjects

The present invention can be applied to any subject suffering fromcolorectal cancer or at risk to develop colorectal cancer. Inparticular, the therapeutic effect of said complex may be to elicit animmune response directed against said antigens or antigenic epitopes, inparticular a response that is dependent on CD4+ helper T cells and/orCD8+ cytotoxic T cells and/or that is restricted by MHC class Imolecules and/or MHC class II molecules.

It is also preferred that subjects according to the invention have beensubjected to a surgical removal of a tumor.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

All references cited herein are herewith incorporated by reference.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE FIGURES

In the following a brief description of the appended figures will begiven. The figures are intended to illustrate the present invention inmore detail. However, they are not intended to limit the subject matterof the invention in any way.

FIG. 1 shows for Example 1 expression of activation marker CD40 by humanblood monocyte-derived dendritic cells (DCs) from one single buffy. TheDCs were stimulated with 300 nM of EDAZ13Mad5, Z13Mad5, Mad5 or 25 ng/mlof LPS during 48 h. Isotype staining for each condition was alsoperformed (isotype is not shown in the FIG. 1) (one experiment).

FIG. 2 shows for Example 1 expression of activation marker CD86 by humanblood monocyte-derived dendritic cells (DCs) from one single buffy. TheDCs were stimulated with 300 nM of EDAZ13Mad5, Z13Mad5, Mad5 or 25 ng/mlof LPS during 48 h. Isotype staining for each condition was alsoperformed (isotype is not shown in the FIG. 2) (one experiment).

FIG. 3 shows for Example 1 expression of activation marker HLADR byhuman blood monocyte-derived dendritic cells (DCs) from one singlebuffy. The DCs were stimulated with 300 nM of EDAZ13Mad5, Z13Mad5, Mad5or 25 ng/ml of LPS during 48 h. Isotype staining for each condition wasalso performed (isotype is not shown in the FIG. 3) (one experiment).

FIG. 4 shows for Example 1 expression of activation marker CD83 by humanblood monocyte-derived dendritic cells (DCs) from one single buffy. TheDCs were stimulated with 300 nM of EDAZ13Mad5, Z13Mad5, Mad5 or 25 ng/mlof LPS during 48 h. Isotype staining for each condition was alsoperformed (isotype is not shown in the FIG. 4) (one experiment).

FIG. 5 shows for Example 2 functional MHC class I-restrictedcross-presentation in a murine in an vitro system using bone marrowderived dendritic cells (BMDCs) and splenocytes from different TCRtransgenic mice. To this end, BMDCs were loaded overnight with 300 nM ofEDAZ13Mad5, EDAMad5 or Mad5. Efficient MHC class I-restrictedpresentation of OVACD8 epitope and gp100 epitope was monitored after 4days with CFSE-labeled OT1 cells and P-Mel cells respectively. EfficientMHC class II-restricted presentation of OVACD4 epitope was monitoredafter 4 days with CFSE-labeled OT2 cells. As control, BMDCs were pulsedfor 1 h with 5 uM peptide (one experiment representative of 2 individualexperiments).

FIG. 6 shows the results for the 2 nmol groups for Example 3. C57BL/6mice were vaccinated twice (Wk0 and Wk2) with 2 nmol of EDAMad5 orEDAZ13Mad5. Positive control group was vaccinated with Mad5 and MPLA(equimolar to EDA). Mice were bled 7 days after last vaccination andpentamer staining was performed (3-4 mice per group, one experiment).

FIG. 7 shows the results for the 10 nmol groups for Example 3. C57BL/6mice were vaccinated twice (Wk0 and Wk2) with 10 nmol of EDAMad5 orEDAZ13Mad5. Positive control group was vaccinated with Mad5 and MPLA(equimolar to EDA). Mice were bled 7 days after last vaccination andpentamer staining was performed (3-4 mice per group, one experiment).

FIG. 8 shows for Example 3 the percentage of pentamer positive CD8+ Tcells for all groups tested. C57BL/6 mice were vaccinated twice (Wk0 andWk2) with 2 nmol or 10 nmol of EDAMad5 or EDAZ13Mad5. Positive controlgroup was vaccinated with Mad5 and MPLA (equimolar to EDA). Mice werebled 7 days after last vaccination and pentamer staining was performed(one experiment with 3-4 mice per group).

FIG. 9 shows for Example 4 the tumor growth of 7 mice per group(mean±SEM); *, p<0.05 EDAZ13Mad5 versus control group (2-way Anovatest). C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cellsin the left flank and vaccinated twice (d5 and d13) by subcutaneousinjection of 10 nmol of EDAZ13Mad5, EDAMad5, Mad5 or Mad5 and MPLA(equimolar to EDA) s.c. in the right flank. Tumor size was measured witha caliper.

FIG. 10 shows for Example 4 individual tumor growth curves (7 individualmice per group). C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVAtumor cells in the left flank and vaccinated twice (d5 and d13) bysubcutaneous injection of 10 nmol of EDAZ13Mad5, EDAMad5, Mad5 or Mad5and MPLA (equimolar to EDA) s.c. in the right flank. Tumor size wasmeasured with a caliper.

FIG. 11A-B shows for Example 4 (A) the survival curve of 7 mice pergroup; *, p<0.05 EDAZ13Mad5 versus control group (Log-rank test) and (B)the tumor-free progression curve of 7 mice per group; *, p<0.05EDAZ13Mad5 versus control group (Log-rank test).

FIG. 12 shows for Example 5 the number of metastasis for everyexperimental group. C57BL/6 mice were implanted i.v. with 1×10⁵ B16-OVAmelanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneousinjection of 2 nmol of EDAZ13Mad5, EDAMad5 or Z13Mad5+ MPLA (equimolarto EDA) or MPLA alone s.c. in the right flank. Mice were euthanized atday 13 and lung recovered. Number of metastasis foci was counted foreach lung. **, p<0.01; ****, p<0.0001 (Unpaired T test).

FIG. 13 shows for Example 6 the number of metastasis for everyexperimental group. C57BL/6 mice were vaccinated twice (d-21 and d-7) bysubcutaneous injection of 2 nmoles of EDAZ13Mad5, EDAMad5 or Z13Mad5+MPLA (equimolar to EDA) s.c. in the right flank. At day 0, mice wereimplanted i.v. with 1×10⁵ B16-OVA melanoma tumor cells. Mice wereeuthanized at day 14 and lung recovered. Number of metastasis foci wascounted for each lung. *, p<0.05. ***, p<0.001 (Unpaired T test).

FIG. 14A-B shows the results for Example 8. HEK-hTLR2 cell lines wereseeded in flat 96-well plate in culture medium, stimulated with 0.3 μM,1 μM or 3 μM of AnaxaZ13Mad5 or Z13Mad5Anaxa and incubated at 37° C. for24 h. Positive control was performed with 500 ng/ml of Pam3CSK4. (A)Twenty microliters of supernatant were added to QuantiBlue® detectionmedium and incubated at 37° C. for 1 h before OD reading (620 nm). (B)Quantification of IL-8 secretion (by ELISA) in the supernatant.

FIG. 15 shows the results for Example 9. C57BL/6 mice were vaccinatedtwice (Wk0 and Wk2) with 2 nmoles of Z13Mad5Anaxa or AnaxaZ13Mad5. Micewere bled 7 days after last vaccination and pentamer staining wasperformed (one experiment).

FIG. 16 shows the results for Example 9. C57BL/6 mice were vaccinatedtwice (Wk0 and Wk2) with 2 nmoles Z13Mad5Anaxa or AnaxaZ13Mad5. Micewere bled 7 days after last vaccination and pentamer staining wasperformed (one experiment with 4 mice per group). *, p<0.05.

FIG. 17 shows for Example 10 the tumor growth of 7 mice per group(mean±SEM). C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumorcells in the left flank and vaccinated twice (d5 and d13) bysubcutaneous injection of 10 nmol of either AnaxZ13Mad5, Z13Mad5Anaxa orco-injection of Z13Mad5+ Pam3CSK4 (equimolar to Anaxa) in the rightflank. Tumor size was measured with a caliper. *, p<0.05; ***, p<0.0001,****, p<0.0001.

FIG. 18 shows for Example 10 the individual tumor growth curves (7individual mice per group). C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13)by subcutaneous injection of 10 nmol of either AnaxZ13Mad5, Z13Mad5Anaxaor co-injection of Z13Mad530 Pam3CSK4 (equimolar to Anaxa) s.c. in theright flank. Tumor size was measured with a caliper.

FIG. 19 shows for Example 10 the survival curve of 7 mice per group.C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells in theleft flank and vaccinated twice (d5 and d13) by subcutaneous injectionof 10 nmol of either AnaxZ13Mad5, Z13Mad5Anaxa or co-injection ofZ13Mad5+ Pam3CSK4 (equimolar to Anaxa) in the right flank. Tumor sizewas measured with a caliper. *, p<0.05, **, p<00.1, ****, p<0.0001(Log-rank test).

FIG. 20 shows for Example 11 the tumor growth of 7 mice per group(mean±SEM). C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumorcells in the left flank and vaccinated twice (d5 and d13) bysubcutaneous injection of 2 nmoles of Hp91Z13Mad5, EDAZ13Mad5,Z13Mad5Anaxa, Z13Mad5EDA or Z13Mad5 and MPLA (equimolar to EDA) in theright flank. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 (2-wayAnova test at day 23).

FIG. 21 shows for Example 11 the individual tumor growth curves (7individual mice per group). C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13)by subcutaneous injection of 2 nmoles of Hp91Z13Mad5, EDAZ13Mad5,Z13Mad5Anaxa, Z13Mad5EDA or Z13Mad5 and MPLA (equimolar to EDA) s.c. inthe right flank.

FIG. 22 shows for Example 11 the survival curves of all 7 mice pergroup. Median survival is indicated on the graph (m.s.). *, p<0.05; **,p<0.01 (Log-rank test).

FIG. 23 shows for Example 12 the tumor growth of 7 mice per group(mean±SEM); ****, p<0.0001 (Log-rank test). C57BL/6 mice were implanteds.c. with 3×10⁵ EG7-OVA tumor cells in the left flank and vaccinatedtwice (once at d5 and once at d13) by subcutaneous injection of either0.5 nmol, 2 nmol or 10 nmol of Z13Mad5Anaxa in the right flank. Tumorsize was measured with a caliper.

FIG. 24A-B shows for Example 13 the SIINFEKL-specific CD8 T cellresponses detected in the blood of C57BL/6 mice vaccinated three times(once at Wk0, once at Wk2 and once at Wk4) s.c., i.d. or i.m. with 0.5nmol (A) or 2 nmol (B) of Z13Mad5Anaxa. Blood was obtained from mice 7days after the 2nd and the 3rd vaccination and multimer staining wasperformed (one experiment with 4 mice per group). *, p<0.05.

FIG. 25A-B shows for Example 13 KLRG1 expression (A) and PD-1 expression(B), which were analyzed on multimer-positive CD8 T cells (oneexperiment with 4 mice per group). Briefly, C57BL/6 mice were vaccinatedthree times (once at Wk0, once at Wk2 and once at Wk4) s.c., i.d. ori.m. with 2 nmol of Z13Mad5Anaxa. Blood was obtained from mice 7 daysafter the 2nd and the 3rd vaccination and FACS staining was performed.

FIG. 26 shows for Example 14 SIINFEKL-specific CD8 T cell responses inC57BL/6 mice vaccinated two times (once at Wk0 and once at Wk2)intranodally with 0.5 nmol of Z13Mad5Anaxa. Blood was obtained from mice7 days after the 2nd vaccination and multimer staining was performed (3mice per group).

FIG. 27A-D shows for Example 15 the percentage of pentamer-positivecells among CD8 T cells (A and B; *, p<0.05) and KLRG1 geomean ofpentamer-positive CD8 T cells (C and D). Briefly, C57BL/6 mice werevaccinated 3 times (A and C: Wk0, Wk2 and Wk4; B and D: Wk0, Wk2 andWk8) s.c. with 2 nmol of Z13Mad5Anaxa. Mice were bled 7 days after lastvaccination and pentamer staining was performed (one experiment with 4mice per group).

FIG. 28A-F shows for Example 15 the percentage of multimer-positivecells among CD8 T cells (A and D); KLRG1 geomean of multimer-positiveCD8 T cells (B and E) and PD1 geomean of multimer-positive CD8 T cells(C and F). A-C, C57BL/6 mice were vaccinated 3 times at Day0, Day3 andDay7 and bled at Day7 and Day14. D-F, C57BL/6 mice were vaccinated 3times at Day0, Day7 and Day14 and bled at Day14 and Day21. Vaccinationwas performed s.c. with 0.5 nmol of Z13Mad5Anaxa. Multimer staining wasperformed on blood samples (one experiment with 4 mice per group).

FIG. 29 shows for Example 16 the IL-6 secretion indicating the APCactivation after incubation of BMDCs with various constructs asindicated in the Figure. Briefly, BMDCs were seeded in flat 96-wellplate in culture medium, stimulated with 1 μM of Z13Mad5Anaxa,Mad5Anaxa, Z13Mad5, EDAZ13Mad5 or EDAMad5 and incubated for 24 h at 37°C. IL-6 secretion was quantified by ELISA in the supernatant. Mean±SEMof 2 to 3 individual experiments.

FIG. 30 shows for Example 16 the TNF-α secretion indicating the APCactivation after incubation of Raw 264.7 cells with various constructsas indicated in the Figure. Briefly, Raw 264.7 cells were seeded in flat96-well plate in culture medium, stimulated with 1 μM of Z13Mad5Anaxa,Mad5Anaxa or Z13Mad5 and incubated for 24 h at 37° C. TNF-a secretionwas quantified by ELISA in the supernatant. Mean±SEM of 2 to 3individual experiments.

FIG. 31 shows for Example 17 the IL-8 secretion indicating TLR4 bindingafter incubation of HEK-hTLR4 cells with various constructs as indicatedin the Figure. Briefly, HEK-hTLR4 were seeded in flat 96-well plate inculture medium, stimulated with 1 μM of Z13Mad5Anaxa, Mad5Anaxa,Z13Mad5, EDAZ13Mad5 or EDAMad5 and incubated 24 h at 37° C. IL-8secretion was quantified by ELISA in the supernatant. Mean±SEM of 2individual experiments.

FIG. 32 shows for Example 18 the number of metastasis in a lungmetastasis model with semitherapeutic settings. Briefly, C57BL/6 micewere implanted i.v. with 1×10⁵ B16-OVA melanoma tumor cells andvaccinated twice (d0 and d9) by subcutaneous injection of 2 nmol ofEDAZ13Mad5, Z13Mad5+ MPLA (equimolar to EDA) or MPLA alone s.c. in theright flank. Mice were euthanized at day 13 and lung recovered. Numberof metastasis foci was counted for each lung. **, p<0.01 (One-way Anovawith Tukey's multiple comparisons test).

FIG. 33 shows for Example 19 the number of metastasis in a lungmetastasis model with semitherapeutic settings. Briefly, C57BL/6 micewere implanted i.v. with 1×10⁵ B16-OVA melanoma tumor cells andvaccinated twice (d0 and d9) by subcutaneous injection of 0.5 nmol ofZ13Mad5Anaxa, Mad5Anaxa or Z13Mad5+ Pam3CSK4 (equimolar to Anaxa) s.c.in the right flank. Mice were euthanized at day 21 and lung recovered.Number of metastasis foci was counted for each lung. *, p<0.05; **,p<0.01 (Unpaired t-test).

FIG. 34 shows for Example 20 the quantification of SIINFEKL-specific CD8T cells in a Quad-Gl261 glioblastoma model. Briefly, C57BL/6 mice wereimplanted i.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinated twice(d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmol ofZ13Mad5 and 2 nmol of Anaxa. SIINFEKL-specific CDS T cells werequantified in blood and in BILs at d28 by multimer staining (5-8 miceper group).

FIG. 35 shows for Example 20 the cytokine secretion. Briefly, C57BL/6mice were implanted i.c. with 5×10⁵ Gl261-Quad tumor cells andvaccinated twice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxaor 2 nmol of Z13Mad5 and 2 nmol of Anaxa. BILs were isolated andcultured during 6 h with matured BMDCs loaded or not with SIINFEKLpeptide in presence of BrefeldinA before intracellular staining forcytokines. % of CD8 T cells secreting cytokine (5-8 mice per group).

FIG. 36 shows for Example 21 the effect of Z13Mad5Anaxa on survival inthe Quad-Gl261 glioblastoma model. Briefly, C57BL/6 mice were implantedi.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinated three times (d7,d21 and d35) by s.c. injection of 2 nmol of Z13Mad5Anaxa. Mice wereweight daily and euthanized when weight loss reached more than 15%.

FIG. 37A-B shows for Example 22 the effect of Z13Mad5Anaxa on tumorgrowth and survival in subcutaneous EG7-OVA tumor model in aprophylactic setting. Briefly, C57BL/6 mice were vaccinated twice (d-21and d-7) by s.c. injection of 0.5 nmol of Z13Mad5Anaxa in the rightflank and then implanted at day0 s.c. with 3×10⁵ EG7-OVA tumor cells inthe left flank. Tumor size was measured with a caliper. (A) Tumor growthof 7 mice per group (mean±SEM); ****, p<0.0001 (2-way Anova test at day30). (B) Survival curve of 7 mice per group. Median survival isindicated on the graph (m.s.). ***, p<0.001 (Log-rank test).

FIG. 38A-B shows for Example 23 the effect of Z13Mad5Anaxa on tumorgrowth and survival insubcutaneous B16-OVA tumor model in a therapeuticsetting on an established tumor. Briefly, C57BL/6 mice were implanteds.c. with 1×10⁵ B16-OVA tumor cells in the left flank and vaccinatedtwice (d14 and d21) by s.c. injection of 0.5 nmol of Z13Mad5Anaxa in theright flank. (A) Tumor growth of 7 mice per group (mean±SEM); *, p<0.05(2-way Anova test at day 32). (B) Survival curve of 7 mice per group.Median survival is indicated on the graph (m.s.).

FIG. 39A-B shows for Example 24 the effect of the CPP in Z13Mad5Anaxa ontumor growth and survival in subcutaneous EG7-OVA tumor model. Briefly,C57BL/6 mice were implanted at day0 s.c. with 3×10⁵ EG7-OVA tumor cellsin the left flank and then vaccinated twice (d5 and d13) by s.c.injection of 0.5 nmol of Z13Mad5Anaxa or Mad5Anaxa in the right flank.Tumor size was measured with a caliper. (A) Tumor growth of 7 mice pergroup (mean±SEM); ****, p<0.0001. (B) Survival curve of 7 mice pergroup. Median survival is indicated on the graph (m.s.). **, p<0.01;***, p<0.001.

FIG. 40A-B shows for Example 25 the effect of complexes having differentCPPs on the immune response. C57BL/6 mice were vaccinated five times(Wk0, Wk2, Wk4, Wk6 and Wk8) s.c. with either 2 nmol (A) or 0.5 nmol (B)of Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa. Mice were bled 7 daysafter the 2^(nd), 3^(rd), 4^(th) and 5^(th) vaccination and multimerstaining was performed (one experiment with 4 mice per group). *, p<0.05between vaccinated versus naïve mice at each time point except afterVac2 for Z18Mad5Anaxa-vaccinated mice.

FIG. 41A-C shows for Example 26 the effect of complexes having differentCPPs on CD8 T cells in spleen (A), draining lymph nodes (B) and bonemarrow (C). C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6and Wk8) s.c. with 2 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa. Nine daysafter the 5th vaccination, mice were euthanized, organs recovered andmultimer staining was performed.

FIG. 42A-B shows for Example 26 the effect of complexes having differentCPPs on T cells in spleen (CD8 T cell response (A) and CD4 T cellresponse (B)). C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4,Wk6 and Wk8) s.c. with 2 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa. (A) ninedays after the 5^(th) vaccination, Elispot assay was performed on spleencells stimulated with SIINFEKL OVACD8 peptide. (B) nine days after the5^(th) vaccination, Elispot assay was performed on spleen cellsstimulated with OVACD4 peptide.

FIG. 43 shows for Example 26 the effect of complexes having differentCPPs on CD8 T cell effector function. C57BL/6 mice were vaccinated fivetimes (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c. with 2 nmol of Z13Mad5Anaxa orZ14Mad5Anaxa. Nine days after the 5^(th) vaccination, intracellularstaining was performed on spleen cells stimulated with SIINFEKL OVACD8peptide.

FIG. 44A-B shows for Example 27 the effect of complexes having differentCPPs on tumor growth (A) and survival rates (B). C57BL/6 mice wereimplanted s.c. with 3×10⁵ EG7-OVA tumor cells in the left flank andvaccinated twice (d5 and d13) by s.c. injection of 0.5 nmol ofZ13Mad5Anaxa or Z14Mad5Anaxa in the right flank. (A) Tumor growth of 7mice per group (mean±SEM); *, p<0.05; ****, p<0.0001 (2-way Anova testat day 28). (B) Survival curve of 7 mice per group. Median survival isindicated on the graph (m.s.). *, p<0.05; **, p<0.01; ***,p<0.001(Log-rank test).

FIG. 45A-B shows for Example 28 the effect of complexes having differentCPPs on the immune response. C57BL/6 mice were vaccinated three times(Wk0, Wk2 and Wk4) s.c. with 2 nmol (A) or 0.5 nmol (B) of EDAZ13Mad5,EDAZ14Mad5 or EDAZ18Mad5. Mice were bled 7 days after the 3^(rd)vaccination and multimer staining was performed (one experiment with 4mice per group). *, p<0.05

FIG. 46A-B shows for Example 29 the effect of EDAZ14Mad5 on tumor growth(A) and survival rates (B). C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13)by s.c. injection of 2 nmoles of EDAZ14Mad5 in the right flank. Leftpanel: Tumor growth of 7 mice per group (mean±SEM); **, p<0.01 (2-wayAnova test at day 27). Right panel: Survival curve of 7 mice per group.Median survival is indicated on the graph (m.s.).

FIG. 47A-B shows for Example 30 the quantification of SIINFEKL-specificCD8 T cells in a Quad-Gl261 glioblastoma model. Briefly, C57BL/6 micewere implanted i.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinatedtwice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmolof Z13Mad5 and 2 nmol of Anaxa. SIINFEKL-specific CD8 T cells werequantified in blood (A) and in BILs (B) at d28 by multimer staining(7-16 mice per group).

FIG. 48 shows for Example 30 the cytokine secretion. Briefly, C57BL/6mice were implanted i.c. with 5×10⁵ Gl261-Quad tumor cells andvaccinated twice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxaor 2 nmol of Z13Mad5 and 2 nmol of Anaxa. BILs were isolated andcultured during 6 h with matured BMDCs loaded or not with SIINFEKLpeptide in presence of BrefeldinA before intracellular staining forcytokines. % of CD8 T cells secreting cytokine (7-16 mice per group).

FIG. 49A-B shows for Example 31 the effect of Z13Mad8Anaxa on T cells inspleen (CD8 T cell response (A) and CD4 T cell response (B)). C57BL/6mice were vaccinated four times (Wk0, Wk2, Wk4 and Wk6) s.c. with 2 nmolof Z13Mad8Anaxa. (A) one week after the 4^(th) vaccination, Elispotassay was performed on spleen cells stimulated gp70CD8 peptide. (B) oneweek after the 4^(th) vaccination, Elispot assay was performed on spleencells stimulated with gp70CD4 peptide.

FIG. 50A-B shows for Example 32 the effect of Z13Mad11 Anaxa on thenumber of metastasis in the B16 lung metastasis model (A) and on the Tcell response in spleen (B). C57BL/6 mice were vaccinated two times(day0, day10) s.c. with 1 nmol of Z13Mad11Anaxa.

FIG. 51 shows for Example 33 the effect of Z13Mad9Anaxa on T cells inspleen (CD8 T cell response. C57BL/6 mice were vaccinated four times(Wk0, Wk2, Wk4 and Wk6) s.c. with 2 nmol of Z13Mad9Anaxa. One week afterthe 4^(th) vaccination, Elispot assay was performed on spleen cellsstimulated with adpgk peptide.

FIG. 52 shows for Example 34 the effect of complexes having differentCPPs on the immune response. C57BL/6 mice were vaccinated two times (Wk0and Wk2) s.c. with 2 nmol of either Z13Mad5Anaxa or TatFMad5Anaxa. Micewere bled 7 days after the 2^(nd) vaccination and multimer staining wasperformed (one experiment with 8 mice per group).

FIG. 53 shows for Example 35 the quantification of SIINFEKL-specific CD8T cells in naïve mice. Briefly, C57BL/6 mice were vaccinated once (day0)by s.c. injection of 2 nmol of Z13Mad5Anaxa (group “Z13Mad5Anaxa”) or 2nmol of Z13Mad5 and 2 nmol of Anaxa (group “Z13Mad5+Anaxa”).SIINFEKL-specific CD8 T cells were quantified in blood at d7 by multimerstaining (4-8 mice per group).

FIG. 54 shows for Example 36 the effect of Z13Mad12Anaxa on T cells inblood (CD8 T cell response). C57BL/6 mice were vaccinated twice (Wk0 andWk2) s.c. with 2 nmol of Z13Mad12Anaxa. One week after the 2^(nd)vaccination, multimer staining for the neoantigen reps1 was performed onblood cells.

FIG. 55 shows for Example 37 expression of activation marker HLA-DR,CD83, CD80 and CD86 (from left to right) by human blood monocyte-deriveddendritic cells (DCs) from one single buffy. The DCs were stimulatedwith 300 nM of Z13Mad5Anaxa (lower panels) or Z13Mad5 (upper panels)during 48 h. Isotype staining for each condition was also performed asshown.

EXAMPLES

In the following, particular examples illustrating various embodimentsand aspects of the invention are presented. However, the presentinvention shall not to be limited in scope by the specific embodimentsdescribed herein. The following preparations and examples are given toenable those skilled in the art to more clearly understand and topractice the present invention. The present invention, however, is notlimited in scope by the exemplified embodiments, which are intended asillustrations of single aspects of the invention only, and methods whichare functionally equivalent are within the scope of the invention.Indeed, various modifications of the invention in addition to thosedescribed herein will become readily apparent to those skilled in theart from the foregoing description, accompanying figures and theexamples below. All such modifications fall within the scope of theappended claims.

Example 1 In Vitro Human Dendritic Cell Maturation

The goal of this study was to investigate the capacity of a complex foruse according to the present invention to induce maturation of dendriticcells. In the present study, the complex for use according to thepresent invention is a fusion protein, comprising the cell-penetratingpeptide “Z13”, a protein“MAD5”, which consists of different CD8+ andCD4+ epitopes from various antigens, and the TLR4 peptide agonist “EDA”.Accordingly, a fused protein with the EDA peptide at the N-terminalposition and different control conjugated proteins without Z13 or EDA orboth were designed.

Namely, the following constructs were designed, whereby in the aminoacid sequence the cell-penetrating peptide “Z13” is shown underlined andthe TLR peptide agonist “EDA” is shown in italics:

EDAZ13Mad5 Sequence: [SEQ ID NO: 26]MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGEDDTAELQGLR PGSEYTVSVV ALHDDMESQP LIGIQSTKRY KNRVASRKSR AKFKQLLQHY REVAAAKSSE NDRLRLLLKE SLKISQAVHA AHAEINEAGREVVGVGALKV PRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLE SIINFEKLTE WTGS

Molecular weight: 25′057 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains EDA TLR agonist (Lasarte, J. J., et al., The extra        domain A from fibronectin targets antigens to TLR4-expressing        cells and induces cytotoxic T cell responses in vivo. J        Immunol, 2007. 178(2): p. 748-56)    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 1 M L-Arginine, pH 8    -   Endotoxin level: <0.01 EU/ug

Z13Mad5 Sequence: [SEQ ID NO: 29]MHHHHHHKRY KNRVASRKSR AKFKQLLQHY REVAAAKSSENDRLRLLLKE SLKISQAVHA AHAEINEAGR EVVGVGALKVPRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLE SIINFEKLTE WTGS

Molecular weight: 15′196 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 1 M L-Arginine, pH 9    -   Endotoxin level:        -   Batch 1: 0.32 EU/mg        -   Batch 2: 0.44 EU/mg

Mad5 Sequence: [SEQ ID NO: 30] MHHHHHHE SLKISQAVHA AHAEINEAGR EVVGVGALKVPRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLE SIINFEKLTE WTGS

Molecular weight: 10′154.6 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8    -   Endotoxin level: 0.069 EU/mg

The EDAZ13Mad5, Z13Mad5 and Mad5 proteins were investigated for theircapacity to induce human dendritic cell (DC) maturation. Afterincubation during 48 h with 300 nM of protein, activation markersexpression (CD86, CD40, CD83 and HLA-DR) was assessed on the human DCsby FACS (FIGS. 1-4). Specific buffers of each protein were used asnegative controls.

Results are shown for CD40 in FIG. 1, for CD86 in FIG. 2, for HLADR inFIG. 3, and for CD83 in FIG. 4. Whereas EDAZ13Mad5 induced maturation ofhuman DCs, shown by the up-regulation of CD86, HLADR and CD83, Z13Mad5and Mad5 proteins were not able to activate human DCs. These resultsindicate that the EDA portion of the protein is responsible for theup-regulation of the activation markers on the human DCs.

Example 2 In Vitro Epitope Presentation (MHC I)

The goal of this study was to assess functional MHC class I-restrictedcross-presentation in a murine in an vitro system using bone marrowderived dendritic cells (BMDCs) and splenocytes from different TCRtransgenic mice. To this end, the constructs EDAZ13Mad5 and Mad5(described above in Example 1) and the construct EDAMad5 were used:

EDAMad5 Sequence [SEQ ID NO: 31]MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGEDDTAELQGLR PGSEYTVSVVALHDDMESQP LIGIQSTE SLKISQAVHA AHAEINEAGREVVGVGALKV PRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLE SIINFEKLTE WTGS

Molecular weight: 20′017 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains EDA TLR agonist    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8    -   Endotoxin level: 1.8 EU/mg

BMDCs were loaded overnight with 300 nM of with the EDAMad5, EDAZ13Mad5and Mad5 proteins containing OVACD8, OVACD4 and gp100 epitopes.Processing and presentation of these MHC I-restricted OVACD8 and gp100epitopes were monitored by measuring the in vitro proliferation of naïveOVA₂₅₇₋₂₆₄-specific CD8+ T cells from OT-1 T cell receptor (TCR)transgenic mice and gp100-specific CD8+ T cells from P-mel T cell TCRtransgenic mice respectively. Accordingly, efficient MHC classI-restricted presentation of OVACD8 epitope and gp100 epitope wasmonitored after 4 days with CFSE-labeled OT1 cells and P-Mel cellsrespectively. Processing and presentation of MHC II-restricted OVACD4epitope was monitored by measuring the in vitro proliferation of naïveCD4+ T cells from OT-2 T cell receptor (TCR) transgenic mice.Accordingly, efficient MHC class II-restricted presentation of OVACD4epitope was monitored after 4 days with CFSE-labeled OT2 cells. Ascontrol, BMDCs were pulsed for 1 h with 5 uM peptide (one experimentrepresentative of 2 individual experiments).

Results are shown in FIG. 5. Similar cross-presentation and processingcapacity of all assessed Mad5-based proteins were observed.

Example 3 CD8 T Cell Immune Response

To investigate the efficacy of EDA-conjugated proteins in inducingpolyclonal CD8+ T cell response, C57BL/6 mice were vaccinated twice (Wk0and Wk2), by subcutaneous injection of either 2 nmol or 10 nmol of theconstructs EDAZ13Mad5 or EDAMad5 (described in Examples 1 and 2).Positive control group was vaccinated with Mad5 and the TLR4 agonistMPLA (equimolar to EDA). Two doses were assessed 2 nmol of the construct(FIG. 6) and 10 nmol of the construct (FIG. 7). 3-4 mice were used pergroup.

Seven days after the last vaccination, mice were bled and pentamerstaining was performed to monitor the OVA-specific immune response inthe blood. In FIG. 8, the percentage of pentamer positive CD8+ T cellsis shown for all groups and both doses tested.

These data show that interestingly the immune response is lower at 10nmol compared to 2 nmol. At both doses, 2 nmol and 10 nmol, the vaccinemediated immune response was observed more consistently in theEDAZ13Mad5 group in contrast to the EDAMad5 group. Moreover, there is anincreased immune response when the TLR4 agonist is conjugated with thevaccine.

Example 4 Vaccine Efficacy on Tumor Growth in a Benchmark EG.7-OVA TumorModel

To evaluate the effect of EDA construct proteins on tumor growthcontrol, the s.c. model of EG.7-OVA thymoma cells was chosen. C57BL/6mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells in the leftflank. After tumor implantation, mice were vaccinated at day 5 and 13with 10 nmol of one of the following constructs (cf. Examples 1 and 2for construct description): EDAZ13Mad5, EDAMad5, Mad5, or Mad5 and MPLA(equimolar to EDA) s.c. in the right flank. Tumor size was measured witha caliper.

FIG. 9 shows the tumor growth of 7 mice per group (mean±SEM); *, p<0.05EDAZ13Mad5 versus control group (2-way Anova test). FIG. 10 showsindividual tumor growth curves (7 individual mice per group). FIG. 11 Ashows the survival curve of 7 mice per group; *, p<0.05 EDAZ13Mad5versus control group (Log-rank test). FIG. 11 B shows the tumor-freeprogression curve of 7 mice per group; *, p<0.05 EDAZ13Mad5 versuscontrol group (Log-rank test).

The results show that in a therapeutic setting, EDAZ13Mad5 was the onlyprotein vaccine to significantly control the tumor growth compared tothe control group with a significant better tumor free progression curveand survival curve.

The results therefore suggest that the construct protein EDAZ13Mad5 is ahighly potent vaccine for controlling the tumor growth in a therapeuticsetting.

Example 5 Vaccine Efficacy on Tumor Growth in a Melanoma MetastasisModel

To assess the efficacy in a lung metastasis model using B16-OVA tumorcells in a semi-therapeutic setting, different construct proteins wereused: EDAMad5, EDAZ13Mad5, Z13Mad5+ MPLA (cf. Examples 1 and 2 fordesign of the constructs), and MPLA alone. C57BL/6 mice were implantedi.v. with 1×10⁵ B16-OVA melanoma tumor cells and at the same time (d0) 2nmol of the vaccine (EDAMad5, EDAZ13Mad5, Z13Mad5+ MPLA, MPLA alone) wasadministered by subcutaneous injection in the right flank. Nine dayslater, mice were vaccinated a second time with the same dose. Furthercontrol groups were vaccinated with 2 nmol of Z13Mad5 and the TLR4agonist MPLA (equimolar to EDA) or MPLA alone. Mice were euthanized atday 13 and lung recovered. Number of metastasis foci was counted foreach lung. The results are shown in FIG. 12.

The results show that the conjugate EDAZ13Mad5 is as potent as Z13Mad5+MPLA to inhibit tumor metastasis in the lung. Furthermore, EDA-Mad5 isless potent than EDAZ13Mad5, indicating a crucial role of Z13 in vaccineefficacy.

Example 6 Vaccine Efficacy on Tumor Growth in a Melanoma MetastasisModel—Prophylactic Setting

Furthermore, the efficacy of the different construct proteins EDAMad5,EDAZ13Mad5, and Z13Mad5+ MPLA (cf. Examples 1 and 2 for design of theconstructs) was assessed in a lung metastasis model in a prophylacticsetting. C57BL/6 mice were vaccinated 21 and 7 days before implantationof tumor cells (d-21 and d-7) by subcutaneous injection of 2 nmol ofEDAZ13Mad5, EDAMad5 or Z13Mad5+ MPLA (equimolar to EDA) s.c. in theright flank. At day 0, mice were implanted i.v. with 1×10⁵ B16-OVAmelanoma tumor cells. Mice were euthanized at day 14 and lung recovered.Results are shown in FIG. 13.

Example 7 Design of Further Constructs Comprising a TLR2 Peptide Agonist

Herein, the complex for use according to the present invention is againa fusion protein, comprising the cell-penetrating peptide “Z13”, theprotein “MAD5”, which consists of different CD8+ and CD4+ epitopes fromvarious antigens, and the TLR2 peptide agonist “Anaxa”. Accordingly,fused proteins with the Anaxa peptide at the C-terminal or N-terminalposition were designed.

Namely, the following constructs were designed, whereby in the aminoacid sequence the cell-penetrating peptide “Z13” is shown underlined andthe TLR peptide agonist “Anaxa” is shown in italics:

AnaxaZ13Mad5 Sequence: [SEQ ID NO: 27]MHHHHHHSTV HEILCKLSLE GDHSTPPSAY GSVKPYTNFD AEKRYKNRVA SRKSRAKFKQ LLQHYREVAA AKSSENDRLRLLLKESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQDWLGVPRFAKF ASFEAQGALA NIAVDKANLD VEQLESIINF EKLTEWTGS

Molecular weight: 18973 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains the 35-mer peptide of Annexin    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8    -   Endotoxin level: 5.17 EU/mg

Z13Mad5Anaxa Sequence: [SEQ ID NO: 28]MHHHHHHKRYKNRVA SRKSRAKFKQ LLQHYREVAAAKSSENDRLR LLLKESLKIS QAVHAAHAEI NEAGREVVGVGALKVPRNQD WLGVPRFAKF ASFEAQGALA NIAVDKANLDVEQLESIINF EKLTEWTGSS TVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAE

Molecular weight: 18973 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains the 35-mer peptide of Annexin    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8

Endotoxin level: 3.1 EU/mg

Example 8 TLR2 Binding (HEK-hTLR2 Cell Lines)

The goal of this study was to assess whether the Z13Mad5Anaxa andAnaxaZ13Mad5 construct proteins (cf. Example 7 for design of theseconstruct proteins) were able to bind TLR2 as an agonist. HEK-Blue™hTLR2 were seeded in flat 96-well plate in culture medium, stimulatedwith 0.3 μM, 1 μM or 3 μM of AnaxaZ13Mad5 or Z13Mad5Anaxa and incubatedat 37° C. for 24 h. Positive control was performed with 500 ng/ml ofPam3CSK4, a TLR2 agonist.

To monitor the activation of NF-KB/AP1, twenty microliters of thesupernatant were added to QuantiBlue® detection medium and incubated at37° C. for 1 h before OD reading (620 nm). Results are shown in FIG. 14A.

The secretion of IL-8 in the supernatant was quantified by ELISA.Results are shown in FIG. 14 B.

Results (FIG. 14 A, B) showed that Z13Mad5Anaxa and AnaxaZ13Mad5 aresimilarly able to bind to TLR2 in a dose dependent manner.

Example 9 In Vivo Induction of Specific CD8+ T Cells

To investigate the efficacy of the Anaxa-conjugated proteins of Example7 in the induction of CD8+ T cell responses, C57BL/6 mice werevaccinated twice (Wk0 and Wk2), by subcutaneous injection of 2 nmol ofAnaxaZ13Mad5 or 2 nmol of Z13Mad5Anaxa. Seven days after the lastvaccination, mice were bled and to monitor the OVA-specific immuneresponse in the blood, pentamer staining was performed (one experimentwith 4 mice per group). Results are shown in FIGS. 15 and 16.

These data indicate that both, the Z13Mad5Anaxa vaccine and theAnaxaZ13Mad5 construct, elicit a strong immune response.

Example 10 Therapeutic Effect on Tumor Growth

To evaluate the effect of the Anaxa-conjugated construct proteinsdesigned in Example 7 on tumor growth control, a benchmark tumor modelwas used, namely the s.c. implantation of EG.7-OVA thymoma cells.

C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells in theleft flank. After tumor implantation, the three groups of 7 mice eachwere vaccinated s.c. in the right flank at day 5 and 13 by subcutaneousinjection of 10 nmol of either AnaxZ13Mad5 (group 1), Z13Mad5Anaxa(group 2) or Z13Mad5 and Pam3CSK4 (equimolar to Anaxa; group 3). Inorder to compare the effect to a protein mixed with an externaladjuvant, a control group was vaccinated with Z13Mad5 and Pam3CSK4(equimolar to Anaxa). Tumor size was measured with a caliper. Resultsare shown in FIG. 17-19.

In a therapeutic schedule, Z13Mad5Anaxa and AnaxaZ13Mad5 are betterprotein vaccines for controlling tumor growth compared to the controlgroup, i.e. co-injection of Z13Mad5 and Pam3CSK showing a significantbetter survival curve. In particular, Z13Mad5Anaxa and AnaxaZ13Mad5demonstrate significantly higher efficacy than Z13Mad5 administratedseparately with Pam3CSK4. The results therefore suggest that theconstruct proteins Z13Mad5Anaxa and AnaxaZ13Mad5 are promisingconjugate-vaccines for controlling the tumor growth in a therapeuticsetting.

Example 11 Therapeutic Effect on Tumor Growth—Comparison of Constructswith Different TLR Agonists

The goal of this study was to compare the efficacy of the differentconstruct protein vaccines conjugated to different TLR agonist, namelyEDAZ13Mad5 and Z13Mad5Anaxa of Example 1 and 7, on tumor growth control.To this end, C57BL/6 mice were implanted s.c. with 3×10⁵ EG.7-OVAthymoma cells in the left flank as described previously in Example 10.Mice (7 individual mice per group) were vaccinated s.c. in the rightflank at day 5 and 13 with 2 nmol of either EDAZ13Mad5, Z13Mad5Anaxa orco-injection of Z13Mad5+ MPLA (equimolar to EDA).

Results are shown in FIGS. 20, 21 and 22. In this experimental setting,Z13Mad5Anaxa, EDAZ13Mad5, and Z13Mad5+ MPLA were similarly able tosignificantly control tumor growth. Moreover, these data indicate thatZ13Mad5Anaxa is the best construct to significantly control tumor growthand EDAZ13Mad5 was slightly better than Z13Mad5+ MPLA in thisexperimental setting.

Example 12 Dose Effect of Z13Mad5Anaxa on Tumor Growth Control

To identify the optimal dose of the conjugate vaccine, three differentdoses (0.5 nmol, 2 nmol and 10 nmol) of Z13Mad5Anaxa (cf. Example 7)were assessed for their ability to control tumor growth. The dose effectof Z13Mad5Anaxa construct was evaluated in the s.c. model of EG.7-OVAthymoma cells as described previously in Example 10. After tumorimplantation, mice were vaccinated twice (at day 5 and at day 13 aftertumor implantation) in a therapeutic setting at 0.5, 2 or 10 nmol ofZ13Mad5Anaxa.

C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells in theleft flank and vaccinated twice (d5 and d13) by subcutaneous injectionof either 0.5 nmol, 2 nmol or 10 nmol of Z13Mad5Anaxa in the rightflank. Tumor size was measured with a caliper.

The tumor growth of 7 mice per group is depicted in FIG. 23. Those datashow that the doses of 0.5 and 2 nmol are at least as efficacious as 10nmol for controlling tumor growth.

Example 13 Effect of Different Routes of Administration of Z13Mad5Anaxa

This study was based on the previous Examples demonstrating the efficacyof Z13Mad5Anaxa conjugate vaccine (cf. Example 7), which is able toelicit specific immune responses and is efficacious for controllingtumor growth in the subcutaneous tumor model EG7 as shown above.

To investigate the effect of subcutaneous, intramuscular and intradermalroutes of administration, immune responses elicited by subcutaneous,intramuscular and intradermal injection were compared. Intradermalinjections were performed using the PLEASE® device from PantecBiosolutions.

Mice were vaccinated three times every two weeks (Wk0, Wk2 and Wk4) with0.5 or 2 nmol of Z13Mad5Anaxa (cf. Example 7). In order to targetseveral lymph nodes, the 1st and the 3rd vaccinations were performed inthe right flank whereas the 2nd was done in the left flank.SIINFEKL-specific CD8+ T cell response was analyzed 1 week after the 2ndand the 3rd vaccination in the blood. FIG. 24 shows theSIINFEKL-specific CD8 T cell responses after each vaccination detectedin the blood of C57BL/6 mice vaccinated three times (Wk0, Wk2 and Wk4)s.c., i.d. or i.m. with 0.5 nmol (FIG. 24 A) or 2 nmol (FIG. 24 B) ofZ13Mad5Anaxa. Blood was obtained from mice 7 days after the 2nd and the3rd vaccination and multimer staining was performed (one experiment with4 mice per group).

The results indicate that at the two doses assessed (0.5 and 2 nmol),(i) all routes of administration tested elicited a SIINFEKL-specific CD8immune response and (ii) the subcutaneous vaccination elicited thestrongest SIINFEKL-specific CD8 immune response. For subcutaneousadministration, the maximum response was reached after the 3ndvaccination and still maintained after the 3rd vaccination. TheSIINFEKL-specific CD8 immune response after the 2nd vaccination elicitedby intradermal and intramuscular vaccinations is lower compared tosubcutaneous vaccination and is not enhanced after the 3rd vaccination.

Next, the effector function and the exhaustion status ofSIINFEKL-specific CD8 T cells was evaluated by analyzing KLRG 1 (Killercell lectin-like receptor subfamily G member 1) and PD-1 respectively.

To this end, C57BL/6 mice were vaccinated three times (Wk0, Wk2 and Wk4)s.c., i.d. or i.m. with 2 nmol of Z13Mad5Anaxa (cf. Example 7). Bloodwas obtained from mice 7 days after the 2nd and the 3rd vaccination andFACS staining was performed. KLRG1 and PD-1 expression were analyzed onmultimer-positive CD8 T cells (one experiment with 4 mice per group).Results are shown in FIG. 25.

These data indicate that the expression of KLRG 1 is strongly increasingon SIINFEKL-specific CD8 T cells after subcutaneous vaccination. Afteri.d. or i.m. vaccination, the observed effects were lower. Thepercentage of KLRG 1-positive cells among SIINFEKL-specific CD8 T cellsis also enhanced after s.c. vaccination (data not shown).

In contrast to KLRG 1, PD-1 expression is decreasing with the time andthe vaccinations, for subcutaneous and intramuscular vaccination routes.This suggests that SIINFEKL-specific CD8 T cells are not exhausted. Thepercentage of PD1-positive cells among SIINFEKL-specific CD8 T cells isalso reduced after s.c. and i.m. vaccination (data not shown). It isimportant to note that PD-1 expression is higher after the 2ndvaccination when mice were vaccinated subcutaneously, reflecting theearly activation status of specific T cells (Keir, M. E., et al., PD-1and its ligands in tolerance and immunity. Annu Rev Immunol, 2008. 26:p. 677-704).

The expression of the late exhaustion marker Tim-3 was also analyzed. Avery low expression as observed for all groups.

Taken together, results indicate that subcutaneous vaccination elicitsthe best specific CD8 immune response compared to intramuscular orintradermal injections.

Example 14 Intranodal Route of Administration

Based on the previous experiments (Example 13), the intranodal route ofadministration was additionally investigated. To this end, the immuneresponse elicited by intranodal injection of Z13Mad5Anaxa (cf. Example7) was investigated.

For this purpose, mice were first injected with Evans Bluesubcutaneously in order to allow easily visualizing the lymph nodes forinjection and inject intranodally without invasive surgery, for exampleas described in Jewell, C. M., S. C. Lopez, and D. J. Irvine, In situengineering of the lymph node microenvironment via intranodal injectionof adjuvant-releasing polymer particles. Proc Natl Acad Sci USA, 2011.108(38): p. 15745-50.

C57BL/6 mice were vaccinated two times every two weeks (Wk0 and Wk2)intranodally with 0.5 nmol of Z13Mad5Anaxa (cf. Example 7). The 1stvaccination was performed in the right inguinal lymph node, whereas thesecond vaccination was done in the left inguinal lymph node. Blood wasobtained from mice 7 days after the 2nd vaccination and multimerstaining was performed (3 mice per group). In other words,SIINFEKL-specific CD8+ T cell response was analyzed one week after the2nd vaccination in the blood. FIG. 26 shows the SIINFEKL-specific CD8 Tcell responses. Those data indicate that also intranodal injection wasable to elicit SIINFEKL-specific CD8 T cells.

Example 15 Vaccination Schedule

The vaccination schedule evaluation work was initiated with theobjective to identify the impact of the third vaccination using the sameZ13Mad5Anaxa construct as described above (cf. Example 7). Thesubcutaneous route was chosen given the previous results.

In the experiment first two vaccinations were performed at wk0 and wk2with a 3rd vaccination either at wk4 (FIG. 27 A) or at wk8 (FIG. 27B).Thus, C57BL/6 mice were vaccinated three times (FIG. 27 A and C: Wk0,Wk2 and Wk4 and FIG. 27 B and D: Wk0, Wk2 and Wk8) s.c. with 2 nmol ofZ13Mad5Anaxa. Blood was obtained from mice 7 days after last vaccinationand pentamer staining was performed (one experiment with 4 mice pergroup). Accordingly, SIINFEKL-specific CD8+ T cell response was analyzed1 week after the 2nd and the 3rd vaccination (FIGS. 27 A and B).Additionally, the effector function of SIINFEKL-specific T cells wasevaluated by analyzing the expression of KLRG 1 on specific CD8 T cells(FIGS. 27C and D).

The data indicate that compared to control the percentage ofSIINFEKL-specific CD8 T cells was significantly increased at all timepoints tested (Vac2 and Vac3) as well as in both vaccination schedules(FIGS. 27 A and B).

Interestingly, the third vaccination at Wk4 allowed to most prominentlyincreasing the percentage of SIINFEKL-specific CD8 T cells (FIG. 27 A).The same cells also demonstrate an improved effector function throughhigher KLRG 1 expression (FIG. 27 C). In contrast, with a thirdvaccination performed at Wk8 no improvement from the second to the thirdvaccination could be observed in the SIINFEKL-specific immune responseand in the KLRG 1 expression.

Taken together, these results indicate that the CD8 immune responsecould be increased by shorten the delay between the second and the thirdvaccination.

Given that an earlier third vaccination seems to increase immuneresponse, in the next study two short schedules of vaccination wereinvestigated:

i) three vaccinations at day 0, day 3 and day 7 and

ii) three vaccinations at day 0, day 7 and day 14.

Again, C57BL/6 mice were used and vaccination was performed s.c. with0.5 nmol of Z13Mad5Anaxa (cf. Example 7). Multimer staining wasperformed on blood samples obtained one week after the 2nd and the 3rdvaccination (one experiment with 4 mice per group).

Thus, SIINFEKL-specific CD8+ T cell response was analyzed one week afterthe 2nd and the 3rd vaccination (FIGS. 28A and D). Additionally, theeffector function of SIINFEKL-specific T cells was evaluated byanalyzing the expression of KLRG 1 on specific CD8 T cells (FIGS. 28Band 28E) and the exhaustion status by analyzing the PD-1 expression ofspecific T cells (FIGS. 28C and 28F).

The data indicate that—similarly to the first study regarding thevaccination schedule described above—compared to control the percentageof SIINFEKL-specific CD8 T cells was increased at all time points tested(Vac2 and Vac3) as well as in both vaccination schedules (FIGS. 28 A andB).

However, compared to the schedule wk0-wk2-wk4, a schedule withvaccinations at Day0, Day3 and Day7 did not elicit such a highSIINFEKL-specific CD8 T cell immune response. Concerning the schedulewith vaccinations at Day0, Day7 and Day14, the SIINFEKL-specific CD8 Tcell immune response elicited is better compared to the previousschedule (d0-d3-d7) but is not maintained after the 3rd vaccination.

Taken together, vaccination schedule data set indicates that theWk0-Wk2-Wk4 vaccination schedule is the best vaccination schedule forinducing potent OVA-specific CD8 immune response with high effectorfunction.

Example 16 Capacity of TLR Agonist-CPP Conjugate Constructs to ActivateMurine Antigen-Pesenting Cells (APCs)

To investigate the effect of both, the CPP component and the TLR agonistcomponent in a complex for use according to the present invention, againthe fusion proteins as described above (cf. Examples 1, 2 and 7) wereused.

In addition, a further “control peptide” was designed, which is also afusion protein and which comprises the protein “MAD5”, which consists ofdifferent CD8+ and CD4+ epitopes from various antigens, and the TLR2peptide agonist “Anaxa” (i.e. without cell penetrating peptide).Accordingly, the following control construct was additionally designed:

Mad5Anaxa Sequence: [SEQ ID NO: 32]MHHHHHHESL KISQAVHAAH AEINEAGREV VGVGALKVPRNQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESIINFEKLTEWT GSSTVHEILC KLSLEGDHST PPSAYGSVKP YTNFDAE

Molecular weight: 13933 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains the 35-mer peptide of Annexin in C-terminal position    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8    -   Endotoxin level: Batch 1-12.15 EU/mg

The aim of this study was to evaluate the capacity of two exemplarycomplexes according to the present invention, namely EDAZ13Mad5 (cf.Example 1) and Z13Mad5Anaxa (cf. Example 7), to promoteantigen-presenting cells activation in comparison to reference complexeslacking either the cell penetrating peptide component Z13 (Mad5Anaxa,cf. above; EDAMad5, cf. Example 2) or the TLR agonist (Z13Mad5, cf.Example 1).

To this end, the capacity of the above mentioned constructs to promoteantigen-presenting cells (APC) activation was assessed in bonemarrow-derived dendritic cells (BMDCs), which express all TLRs exceptTLR7.

BMDCs were seeded in flat 96-well plate in culture medium, stimulatedwith 1 μM of either Z13Mad5Anaxa (cf. Example 7), Mad5Anaxa (cf. above),Z13Mad5 (cf. Example 1), EDAZ13Mad5 (cf. Example 1) or EDAMad5 (cf.Example 2) and incubated for 24 h at 37° C.

The APC activation was investigated by monitoring the secretion of IL-6in the culture supernatant of BMDCs. IL-6 secretion was quantified byELISA in the supernatant.

The results are shown in FIG. 29. These data clearly show thatZ13Mad5Anaxa was able to activate BMDCs, whereas no such activation wasobserved when the cells were cultured in presence of Z13Mad5 orMad5Anaxa. This suggests that not only the TLR agonist (Anaxa or EDA) iscritical for the activation of macrophages and dendritic cells, but thatthe CPP is also needed. Also the presence of the CPP without the TLRagonist is not sufficient, but indeed both, CPP and TLR agonist arecritical for the activation of macrophages and dendritic cells.

Those results were confirmed by using another cell line, namely in theRaw 264.7 mouse macrophage cell line, which expresses all TLRs exceptTLR5 (Applequist, S. E., R. P. Wallin, and H. G. Ljunggren, Variableexpression of Toll-like receptor in murine innate and adaptive immunecell lines. Int Immunol, 2002. 14(9): p. 1065-74).

Raw 264.7 cells were seeded in flat 96-well plate in culture medium,stimulated with 1 μM of either Z13Mad5Anaxa (cf. Example 7), Mad5Anaxa(cf. above) or Z13Mad5 (cf. Example 1) and incubated for 24 h at 37° C.

In Raw 264.7 cells the APC activation was investigated by monitoring thesecretion of TNF-α in the culture supernatant of Raw 264.7. TNF-αsecretion was quantified by ELISA in the supernatant. The results areshown in FIG. 30.

It is thought that the CPP may facilitate the entry of the molecule intothe cells, allowing a better targeting of intracellular TLR.

Taken together, the data reveal the critical role of both, CPP and TLRagonist, within the conjugate constructs to activate APC. This effectmay be due to helping the entry of the construct into the cells,therefore resulting in an optimal targeting of the intracellular TLR.

Example 17 Ability of the Conjugate Constructs to Bind to Human TLR4

It was recently shown that the Anaxa peptide owns an adjuvant activityby signaling through TLR2 (WO 2012/048190 A1), whereas the EDA peptideis a natural ligand for TLR4 (Okamura, Y., et al., The extra domain Aoffibronectin activates Toll-like receptor 4. J Biol Chem, 2001.276(13): p. 10229-33).

Moreover, as shown above in Example 8 and FIG. 14, a complex for useaccording to the present invention comprising the Anaxa peptide as TLRagonist, for example Z13Mad5Anaxa, is able to bind to human TLR2 and topromote the secretion of IL-8 by HEK-hTLR2 cells (cf. Example 8, FIG.14).

In the present study, the ability of complexes according to the presentinvention comprising either the Anaxa peptide as TLR agonist or the EDApeptide as TLR agonist to bind to human TLR4 was evaluated. To this end,HEK cells transfected with human TLR4 (HEK-hTLR4) were seeded in flat96-well plate in culture medium, stimulated with 1 μM of eitherZ13Mad5Anaxa (cf. Example 7), Mad5Anaxa (cf. above), Z13Mad5 (cf.Example 1), EDAZ13Mad5 (cf. Example 1) or EDAMad5 (cf. Example 2) andincubated for 24 h at 37° C. IL-8 secretion was quantified by ELISA inthe supernatant.

Results are shown in FIG. 31. As expected, incubation of HEK-hTLR4 withEDAZ13Mad5 resulted in remarkable IL-8 secretion, indicating binding ofEDAZ13Mad5 to TLR4. In line with the results obtained in Example 16, theIL-8 secretion of EDAMad5 (without the CPP) was remarkably lower ascompared to EDAZ13Mad5, showing the effect of the presence of a CPP. TheZ13Mad5 construct, which does not comprise a TLR agonist, showed no IL-8secretion, indicating—as expected—a lack of binding to TLR4.

Interestingly, incubation of HEK-hTLR4 with the construct Z13Mad5Anaxaresulted in the most pronounced IL-8 secretion, indicating binding ofZ13Mad5Anaxa to TLR4. This is astonishing, since Anaxa was previouslyhypothesized to be a TLR2 agonist. Again, the same construct but withoutthe CPP (Mad5Anaxa) resulted in remarkably lower IL-8 secretion,confirming the results obtained in Example 16.

Taken together, these data (i) confirm the results obtained in Example16, (ii) confirm that EDA is indeed a TLR4 agonist, and (iii) showsurprisingly that the Anaxa peptide is also a TLR4 agonist (in additionto being a TLR2 agonist, cf. Example 8 and FIG. 14).

Example 18 Vaccine Efficacy on Tumor Growth in a Lung MetastasisModel—Semi-Therapeutic Setting: TLR Agonist EDA

This study is based on Example 6, showing the efficacy of a complex foruse according to the present invention, namely EDAZ13Mad5, in a melanomalung metastasis model in a prophylactic setting (cf. FIG. 13).

In the present study the same lung metastasis model was used as well asthe construct proteins EDAZ13Mad5 and Z13Mad5+ MPLA (cf. Examples 1 and2 for design of the constructs). However, in the semi-therapeuticsetting, C57BL/6 mice were vaccinated at the same time as tumor cellswere implanted (d0) and, for a second time, at nine days afterimplantation (d9). Vaccination was performed by subcutaneous injectionof 2 nmol of EDAZ13Mad5, Z13Mad5+ MPLA (equimolar to EDA) or MPLA s.c.in the right flank. At day 0, mice were implanted i.v. with 1×10⁵B16-OVA melanoma tumor cells and vaccinated twice (d0 and d9) bysubcutaneous injection of 2 nmol of EDAZ13Mad5, Z13Mad5+ MPLA (equimolarto EDA) or MPLA alone s.c. in the right flank. Mice were euthanized atday 13 and lung recovered. Results are shown in FIG. 32.

The results show that EDAZ13Mad5 is slightly more potent than Z13Mad5+MPLA to inhibit the growth of melanoma metastasis. In addition, noadjuvant effect was observed in mice injected with MPLA only.

Both, EDAZ13Mad5 and Z13Mad5+ MPLA, significantly inhibit the growth ofmelanoma metastasis in the lung in prophylactic and semitherapeuticsettings.

Example 19 Vaccine Efficacy on Tumor Growth in a Lung MetastasisModel—Semi-Therapeutic Setting: TLR Agonist Anaxa

This study is based on Example 18 with the same model (semitherapeuticsettings) and experimental schedule. However, the effect of complexesaccording to the present invention comprising the “Anaxa” peptide as TLRagonist were investigated—instead of the EDA TLR agonist as in Example18.

To this end, C57BL/6 mice were implanted i.v. with 1×10⁵ B16-OVAmelanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneousinjection of 0.5 nmol of Z13Mad5Anaxa, Mad5Anaxa or Z13Mad5+ Pam3CSK4(equimolar to Anaxa) s.c. in the right flank. Mice were euthanized atday 21 and the lung was recovered. Number of metastasis foci was countedfor each lung. The results are shown in FIG. 33.

The results show that Z13Mad5Anaxa is sensibly more potent than Z13Mad5+Pam3CSK4 to inhibit the growth of melanoma metastasis. In contrast,Mad5Anaxa was not able to control metastasis growth in the lung,underlining again the importance of CPP.

Altogether, the B16-OVA lung metastasis experiment showed thatZ13Mad5Anaxa was highly efficacious in inhibiting the growth of melanomametastasis in the lung.

Example 20 Vaccine Efficacy in a Glioblastoma Model

In this study, another cancer model was used, namely a glioblastomamodel. Glioma is the most frequent form of primary brain tumors inadults, with glioblastoma multiforme (GBM) being the most lethal. Thistumor is notorious for its highly invasive and aggressive behavior.Currently, the best treatment against GBM is a regimen involving acombination of surgery, chemotherapy and radiotherapy, which has amedian survival period of only 14.6 months. There is an urgent, unmetmedical need for new treatment modalities that improve the prognosis ofglioma patients. T-cell mediated immunotherapy is a conceptuallyattractive treatment option to use in conjunction with existingmodalities for glioma, in particular highly invasive GBM.

The Gl261 glioma is a carcinogen-induced mouse glioma model. This modelrepresents one of the very few brain tumor models developed inimmunocompetent animals, that has growth characteristics similar tohuman GBM (Newcomb, E. and D. Zagzag, The murine GL261 gliomaexperimental model to assess novel brain tumor treatments, in CNS CancerModels, Markers, Prognostic, Factors, Targets, and TherapeuticApproaches, E. G. Van Meir, Editor. 2009, Humana Press: Atlanta. p.227-241; Jacobs, V. L., et al., Current review of in vivo GBM rodentmodels: emphasis on the CNS-1 tumour model. ASN Neuro, 2011. 3(3): p.e00063). Low numbers of intracranially transplanted Gl261 cells formedintracranial tumors in C57BL/6 mice (Zhu, X., et al., Poly-/CLC promotesthe infiltration of effector T cells into intracranial gliomas viainduction of CXCL10 in IFN-alpha and IFN-gamma dependent manners. CancerImmunol lmmunother, 2010. 59(9): p. 1401-9; Zhu, X., et al., Toll likereceptor-3 ligand poly-/CLC promotes the efficacy of peripheralvaccinations with tumor antigen-derived peptide epitopes in murine CNStumor models. J Transl Med, 2007. 5: p. 10). The cells are moderatelyimmunogenic: they are able to elicit tumor-specific immune response atthe tumor site. However, the tumor-specific immune cells are not capableof complete tumor clearance.

Recently, M. Ollin generated a new Gl261 model (Ohlfest, J.R., et al.,Vaccine injection site matters: qualitative and quantitative defects inCDB T cells primed as a function of proximity to the tumor in a murineglioma model. J Immunol, 2013. 190(2): p. 613-20) by transfecting Gl261cell line with the “Quad Cassette” expressing four peptides presented byH-2b class I or II molecules: human gp100₂₅₋₅₅, chicken OVA₂₅₇₋₂₆₄,chicken OVA₃₂₃₋₃₅₉, and mouse I-Eα₅₂₋₆₈. The Quad-Gl261 cell line alsostably expresses luciferase, which allows the follow-up of tumor growthby bioluminescence. The goal of this study was to assess the efficacy ofa complex for use according to the present invention in the Quad-Gl261glioblastoma model.

The effect of a complex for use according to the present invention,namely Z13Mad5Anaxa (cf Example 7) was evaluated in the above describedglioblastoma model. T cell homing at the tumor site was thereforeanalyzed in Gl261-Quad tumor-bearing mice vaccinated twice (Wk1 and Wk3)with Z13Mad5Anaxa vaccine. A group vaccinated with Z13Mad5 and Anaxa(equimolar to Z13Mad5Anaxa) administrated separately was used ascontrol. Briefly, C57BL/6 mice were implanted i.c. (intracranially) with5×10⁵ Gl261-Quad tumor cells and vaccinated twice (at d7 and d21following implantation) by s.c. injection of 2 nmol of Z13Mad5Anaxa(group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa (group 2). At Wk4,the blood and the brain infiltrating leukocytes (BILs) were analyzed,whereby SIINFEKL-specific CD8 T cells were quantified in blood and inBILs at d28 by multimer staining (5-8 mice per group). Results are shownin FIG. 34.

In general, low frequency of SIINFEKL-specific CD8 T cells wasquantified in the blood. However, a higher percentage ofSIINFEKL-specific CD8 T cells was observed in the blood ofZ13Mad5Anaxa-vaccinated mice. In all groups, there was a sensiblystronger accumulation of SIINFEKL-specific CD8 T cells in the BILs.

After two vaccinations with Z13Mad5Anaxa, the frequency ofSIINFEKL-specific cells CD8+ T cells in the BILs was 2-fold higher (24%)than with Z13Mad5+ Anaxa (12%).

Next, cytokine secretion was assessed. To this end, C57BL/6 mice wereimplanted i.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinated twice(d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmol ofZ13Mad5 and 2 nmol of Anaxa. BILs were isolated and cultured during 6 hwith matured BMDCs loaded or not with SIINFEKL peptide in presence ofBrefeldinA before intracellular staining for cytokines. Results areshown in FIG. 35.

Despite heterogeneity, a high level of cytokine secretion was observedfor brain-infiltrating CD8 T cells from mice vaccinated withZ13Mad5Anaxa. These results demonstrate that Z13Mad5Anaxa vaccine wasable to elicit a stronger SIINFEKL specific CD8 T cell immune responsein the brain of tumor-bearing mice with potent effector function.

The results obtained are indicating that Z13Mad5Anaxa is efficacious foreliciting high brain infiltrating SIINFEKL-specific CD8 immune response.Z13Mad5Anaxa is able to promote the secretion of cytokine byantigen-specific CD8 T cells in the brain.

Example 21 Vaccine Efficacy on Survival in the Gl261-Quad GlioblastomaModel

In an independent experiment, the survival of control andZ13Mad5Anaxa-vaccinated mice was monitored. The therapeutic settingswere three consecutive vaccinations with 2 nmol of Z13Mad5Anaxa at day7, 21 and 35, post i.c. tumor implantation.

C57BL/6 mice were implanted i.c. with 5×10⁵ Gl261-Quad tumor cells andvaccinated three times (d7, d21 and d35) by s.c. injection of 2 nmol ofZ13Mad5Anaxa. Mice were weight daily and euthanized when weight lossreached more than 15%. Results are shown in FIG. 36.

The results show that Z13Mad5Anaxa therapeutic vaccination is moreefficacious than the control group with a median survival prolonged by10 days.

Example 22 Vaccine Efficacy in a Subcutaneous Tumor Model—ProphylacticSetting

This study is based on the results obtained in Example 10 as shown inFIGS. 17-19.

To evaluate the effect of the Anaxa-conjugated construct proteinsdesigned in Example 7 on tumor growth control, a benchmark tumor modelwas used, namely the s.c. implantation of EG.7-OVA thymoma cells. Incontrast to Example 10, wherein vaccination was performed on days 5 and13, in the present study a prophylactic setting was evaluated, whereinmice were vaccinated 21 and 7 days before tumor implantation.

C57BL/6 mice were vaccinated twice (d-21 and d-7) by s.c. injection of0.5 nmol of Z13Mad5Anaxa in the right flank and then implanted at day0s.c. with 3×10⁵ EG7-OVA tumor cells in the left flank and. Tumor sizewas measured with a caliper.

The results are shown in FIG. 37 with tumor volume (FIG. 37A) andsurvival rate (FIG. 37B). The data is showing that prophylacticvaccination with Z13Mad5Anaxa is highly efficacious for controllingtumor growth and survival rate. The volume of the tumor is highlysignificantly decreased in mice treated with Z13Mad5Anaxa as compared tocontrol mice. The survival rate is highly significantly increased inmice treated with Z13Mad5Anaxa as compared to control mice.

Example 23 Vaccine Efficacy in a Subcutaneous Tumor Model—TherapeuticSetting with Established Tumor

This study is based on the results obtained in Example 10 as shown inFIGS. 17-19 and on the results obtained in Example 22 shown in FIG. 37.It was the goal of this study to evaluate the effect of Z13Mad5Anaxa(cf. Example 7) on an established tumor.

For this purpose, the s.c. model of B16-OVA melanoma cells was used. Inthis model tumor cells are spreading slowly, therefore allowing a biggervaccination time window.

The first vaccination with the low dose of 0.5 nmol of Z13Mad5Anaxa wasperformed once the tumor was established and visible i.e. at day 14after tumor cell implantation. A second vaccination was done at day 21.

Thus, C57BL/6 mice were implanted s.c. with 1×10⁵ B16-OVA tumor cells inthe left flank and vaccinated twice (d14 and d21) by s.c. injection of0.5 nmol of Z13Mad5Anaxa in the right flank. Tumor growth and survivalcurves were monitored. Results are shown in FIG. 38.

The results indicate that Z13Mad5Anaxa efficaciously controls the growthof an established and visible tumor. Moreover, despite an establishedand visible tumor survival rates increased in mice treated withZ13Mad5Anaxa as compared to controls.

Example 24 Vaccine Efficacy in a Subcutaneous Tumor Model—TherapeuticSetting: Effect of the CPP

The protocol of this study corresponds to the study described in Example10, with the difference that an additional group “Mad5Anaxa” (cf.Example 16) was evaluated.

Briefly, a benchmark tumor model was used, namely the s.c. implantationof EG.7-OVA thymoma cells. C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank. After tumor implantation, groupsof 7 mice each were vaccinated s.c. in the right flank at day 5 and 13by subcutaneous injection of 0.5 nmol of either Z13Mad5Anaxa (group 1)or Mad5Anaxa (group 2) and compared to a control group. Tumor size wasmeasured with a caliper. Results are shown in FIG. 39.

The results show that the mice treated with Z13Mad5Anaxa show asignificantly decreased tumor volume and a significantly increasedsurvival rate compared to both, control mice and mice treated withMad5Anaxa, i.e. a construct without CPP. These results indicate that thepresence of a CPP results in significantly decreased tumor volume and asignificantly increased survival rate, i.e. in increased efficiency ofvaccination. Therefore, the results indicate—together with the resultsobtained in Example 10—that the presence of a CPP and the TLR agonistexert a synergic effect on tumor growth and survival rate.

Example 25 Comparison of the Kinetic of Immune Responses with ComplexesHaving Different Cell Penetrating Peptides

To investigate the effect of different CPPs in the complex for useaccording to the present invention the fusion protein Z13Mad5Anaxa asdescribed above (cf. Example 7) was used.

In addition, further fusion proteins were designed, which comprise CPPsother than Z13—namely Z14 (SEQ ID NO: 7) or Z18 (SEQ ID NO: 11). Thosefusion proteins also comprise the protein “MAD5”, which consists ofdifferent CD8+ and CD4+ epitopes from various antigens, and the TLR2peptide agonist “Anaxa”. Accordingly, the following constructs wereadditionally designed:

Z14Mad5Anaxa Sequence: (SEQ ID NO: 33)MHHHHHHKRY KNRVASRKSR AKFKQLLQHY REVAAAKESLKISQAVHAAH AEINEAGREV VGVGALKVPR NQDWLGVPRFAKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWTGSSTVHEILC KLSLEGDHST PPSAYGSVKP YTNFDAE Z18Mad5Anaxa Sequence:(SEQ ID NO: 34) MHHHHHHREV AAAKSSENDR LRLLLKESLK ISQAVHAAHAEINEAGREVV GVGALKVPRN QDWLGVPRFA KFASFEAQGALANIAVDKAN LDVEQLESII NFEKLTEWTG SSTVHEILCK LSLEGDHSTP PSAYGSVKPY TNFDAE

C57BL/6 mice were assigned to eight different groups (4 mice per group):three groups receiving 2 nmol of either Z13Mad5Anaxa, Z14Mad5Anaxa orZ18Mad5Anaxa and a respective control and three groups receiving 0.5nmol of Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa and a respectivecontrol. The mice were vaccinated five times (Week0, Week2, Week4, Week6and Week8) s.c. . . . Mice were bled 7 days after the 2^(nd), 3^(rd),4^(th) and 5^(th) vaccination and multimer staining was performed (oneexperiment with 4 mice per group).

The results are shown in FIG. 40. All groups vaccinated withZ13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa showed an increasedpercentage of multimer-positive cells compared to the control group(except for the second vaccination of Z18Mad5Anaxa). These resultsindicate that complexes according to the present invention havingdifferent cell penetrating peptides are able to elicit an immuneresponse at different doses.

Example 26 Comparison of T Cell Immune Responses with Complexes HavingDifferent Cell Penetrating Peptides

To investigate the CD8 T cell immune responses in more detail, C57BL/6mice were assigned to three different groups (3-4 mice per group):naïve, Z13Mad5Anaxa or Z14Mad5Anaxa. C57BL/6 mice of the Z13Mad5Anaxagroup and of the Z14Mad5Anaxa group were vaccinated five times (Week0,Week2, Week4, Week6 and Week8) s.c. with 2 nmol of either Z13Mad5Anaxa(cf. Example 7) or Z14Mad5Anaxa (cf. Example 25). Nine days after the5^(th) vaccination, mice were euthanized, organs recovered and multimerstaining was performed to identify the percentage of SIINFEKL-specificCD8 T cells in the spleen, bone marrow and draining lymph nodes(inguinal and axillary).

The results are shown in FIG. 41. Mice vaccinated with Z13Mad5Anaxa orwith Z14Mad5Anaxa showed a similar increase in multimer-positive cells,in particular in the spleen and bone marrow as well as a slight increasein draining lymph nodes.

To further investigate the CD8 T cell effector function aftervaccination with complexes with different CPPs, in the same groups ofmice as described above Elispot assay was performed on spleen cellsstimulated with SIINFEKL OVACD8 peptide (SEQ ID NO: 35) nine days afterthe 5^(th) vaccination in order to quantify IFN-γ producing cells.

The results are shown in FIG. 42A. Mice vaccinated with Z13Mad5Anaxashowed a significant increase in IFN-γ producing cells compared to naïvemice. Mice vaccinated with Z14Mad5Anaxa showed also an increase in IFN-γproducing cells compared to naïve mice, however, the increase was notsignificant, which may be due to the low number of mice (3 mice inZ14Mad5Anaxa group).

To investigate the CD4 T cell responses after vaccination with complexeswith different CPPs, in the same groups of mice as described aboveElispot assay was performed on spleen cells stimulated with OVACD4peptide (SEQ ID NO: 36) nine days after the 5^(th) vaccination in orderto quantify IFN-γ producing cells.

The results are shown in FIG. 42B. Mice vaccinated with Z13Mad5Anaxashowed a highly significant increase in IFN-γ producing cells comparedto naïve mice. Mice vaccinated with Z14Mad5Anaxa showed also an increasein IFN-γ producing cells compared to naïve mice, however, the increasewas not significant, which may be due to the low number of mice (3 micein Z14Mad5Anaxa group).

In addition, in the above described groups of mice, intracellularstaining was performed on spleen cells stimulated with SIINFEKL OVACD8peptide (SEQ ID NO: 35) to identify CD107a+IFN-γ+TNF-α+ cells. Resultsare shown in FIG. 43. Mice vaccinated with Z13Mad5Anaxa or withZ14Mad5Anaxa showed a similar increase in CD 107a+IFN-γ+TNF-α+ cells.

Example 27 Comparison of the Effect of Complexes Having Different CellPenetrating Peptides on Tumor Growth and Survival in the EG.7-OVA s.c.Model

To investigate the effects of complexes having different cellpenetrating peptides on tumor growth and survival the EG.7-OVA s.c.model was used. On d0 C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank and assigned to three differentgroups (naïve, Z13Mad5Anaxa and Z14Mad5Anaxa). Mice were vaccinatedtwice at d5 and d13 after tumor implantation by s.c. injection of either0.5 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa in the right flank.

Results are shown in FIG. 44. Vaccination with Z13Mad5Anaxa or withZ14Mad5Anaxa resulted in significantly decreased tumor volumes comparedto control mice (FIG. 44A) as well as to significantly increasedsurvival rates compared to control mice (FIG. 44B). Those resultsindicate that both complexes, Z13Mad5Anaxa and Z14Mad5Anaxa, are able tosignificantly decrease tumor growth and to significantly prolongsurvival.

Example 28 Comparison of the Immune Responses After Vaccination withComplexes Having Different Cell Penetrating Peptides

In this experiment the effect of different CPPs in the complex for useaccording to the present invention was investigated by using a complexwith the TLR agonist “EDA”. Therefore, the fusion protein EDAZ13Mad5 asdescribed above (cf. Example 1) was used.

In addition, further fusion proteins were designed, which comprise CPPsother than Z13—namely Z14 (SEQ ID NO: 7) or Z18 (SEQ ID NO: 11). Thosefusion proteins also comprise the protein “MAD5”, which consists ofdifferent CD8+ and CD4+ epitopes from various antigens, and the TLR4peptide agonist “EDA”. Accordingly, the following constructs wereadditionally designed:

EDAZ14Mad5 Sequence: (SEQ ID NO: 37)MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGE DDTAELQGLR PGSEYTVSVVALHDDMESQP LIGIQSTKRY KNRVASRKSR AKFKQLLQHYREVAAAKESL KISQAVHAAH AEINEAGREV VGVGALKVPRNQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWT GS EDAZ18Mad5Sequence: (SEQ ID NO: 38) MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGE DDTAELQGLR PGSEYTVSVVALHDDMESQP LIGIQSTREV AAAKSSENDR LRLLLKESLKISQAVHAAHA EINEAGREVV GVGALKVPRN QDWLGVPRFAKFASFEAQGA LANIAVDKAN LDVEQLESII NFEKLTEWTG S

C57BL/6 mice were assigned to eight different groups (4 mice per group):three groups receiving 2 nmol of either EDAZ13Mad5, EDAZ14Mad5 orEDAZ18Mad5 and a respective control and three groups receiving 0.5 nmolof either EDAZ13Mad5, EDAZ14Mad5 or EDAZ18Mad5 and a respective controlgroup. The mice were vaccinated three times (Week0, Week2 and Week4)s.c. . . . Mice were bled 7 days after the 2^(nd) and 3^(rd) vaccinationand multimer staining was performed (one experiment with 4 mice pergroup).

The results are shown in FIG. 45. All groups vaccinated with EDAZ13Mad5,EDAZ14Mad5 or EDAZ18Mad5 showed an increased percentage ofmultimer-positive cells compared to the control group. These resultsindicate that complexes according to the present invention havingdifferent cell penetrating peptides are able to elicit an immuneresponse at different doses.

Example 29 Effect of EDAZ14Mad5 on Tumor Growth and Survival in theEG.7-OVA s.c. Model

To investigate the effect of EDAZ14Mad5 on tumor growth and survival theEG.7-OVA s.c. model was used (cf. Example 4 and FIGS. 9-11 for theeffect of EDAZ13Mad5 in the same model).

On d0 C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells inthe left flank and assigned to two different groups (naïve andEDAZ14Mad5). Mice were vaccinated twice at d5 and d13 after tumorimplantation by s.c. injection of 0.5 nmol of EDAZ14Mad5 in the rightflank.

Results are shown in FIG. 46. Similarly to EDAZ13Mad5 (cf. Example 4,FIGS. 9-11) vaccination with EDAZ14Mad5 resulted in significantlydecreased tumor volumes compared to control mice (FIG. 46A) as well asto significantly increased survival rates compared to control mice (FIG.46B). Those results indicate that EDAZ14Mad5 is able to significantlydecrease tumor growth and to significantly prolong survival—similarly toEDAZ13Mad5 (cf. Example 4, FIGS. 9-11).

Example 30 Superior Efficacy of Z13Mad5Anaxa Fusion Construct Comparedto Z13Mad5 and Anaxa in a Glioblastoma Model

To investigate the efficacy of a complex according to the presentinvention the glioblastoma model was chosen (cf. Example 20). Namely,Z13Mad5Anaxa (cf. Example 7; SEQ ID NO: 28) was administered to onegroup of mice, whereas Z13Mad5 (SEQ ID NO: 29) and Anaxa (SEQ ID NO: 15)were administered (both together) to another group of mice.

T cell homing at the tumor site was analyzed in Gl261-Quad tumor-bearingmice (7-16 mice per group) vaccinated twice, namely at day 7 and at day21 after tumor implantation (day 0), with 2 nmol Z13Mad5Anaxa vaccine. Agroup vaccinated with both, Z13Mad5 and Anaxa (equimolar toZ13Mad5Anaxa), was used as control. Briefly, C57BL/6 mice were implantedi.c. (intracranially) with 5×10⁵ Gl261-Quad tumor cells and vaccinatedtwice (at d7 and d21 following implantation) by s.c. injection of 2 nmolof Z13Mad5Anaxa (group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa(group 2). At day 28, the blood and the brain infiltrating leukocytes(BILs) were analyzed, whereby SIINFEKL-specific CD8 T cells werequantified in blood and in BILs at d28 by multimer staining (7-16 miceper group).

Results are shown in FIG. 47. A significantly higher percentage ofSIINFEKL-specific CD8 T cells was observed in the blood ofZ13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with both,Z13Mad5 and Anaxa (FIG. 47A). Similarly, a stronger accumulation ofSIINFEKL-specific CD8 T cells was observed in the BILs ofZ13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with Z13Mad5and Anaxa separately (FIG. 47B, p=0.0539).

Next, cytokine secretion was assessed. To this end, C57BL/6 mice wereimplanted i.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinated twice(d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmol ofZ13Mad5 and 2 nmol of Anaxa. BILs were isolated and cultured during 6 hwith matured BMDCs loaded or not with SIINFEKL peptide (SEQ ID NO: 35)in presence of BrefeldinA before intracellular staining for cytokines.

Results are shown in FIG. 48. In general, a high level of cytokinesecretion was observed for brain-infiltrating CD8 T cells from micevaccinated with Z13Mad5Anaxa. In particular, a significantly highersecretion of total IFN-γ and of IFN-γ and TNF-α together was observedfor brain-infiltrating CD8 T cells from mice vaccinated withZ13Mad5Anaxa as compared to mice vaccinated with Z13Mad5 and Anaxaseparately.

Taken together, these results demonstrate that Z13Mad5Anaxa vaccine (ascompared to Z13Mad5 and Anaxa administered separately) was able toelicit a stronger SIINFEKL specific CD8 T cell immune response in thebrain of tumor-bearing mice with potent effector function.

The results obtained are indicating that Z13Mad5Anaxa is efficacious foreliciting high brain infiltrating SIINFEKL-specific CD8 immune response.Z13Mad5Anaxa is able to promote the secretion of cytokine byantigen-specific CD8 T cells in the brain.

Example 31 Effect of Another Antigenic Cargo in the Complex According tothe Present Invention

To investigate the effect of a different antigenic cargo (“Mad8”),another complex comprising a cell penetrating peptide, differentantigens and a TLR peptide agonist was designed (“Z13Mad8Anaxa”).Z13Mad8Anaxa differs from Z13Mad5Anaxa (described in Example 7) in theantigenic cargoes. In particular, “Z13Mad8Anaxa” is a fusion proteincomprising the cell-penetrating peptide “Z13”, the antigenic cargo“MAD8” comprising CD8 and CD4 epitopes of glycoprotein 70, and the TLRpeptide agonist “Anaxa”. In the following, the amino acid sequence ofZ13Mad8Anaxa is shown with the cell-penetrating peptide “Z13” shownunderlined and the TLR peptide agonist “Anaxa” shown in italics:

(SEQ ID NO: 39) KRYKNRVASR KSRAKFKQLL QHYREVAAAK SSENDRLRLLLKVTYHSPSYAY HQFERRAILN RLVQFIKDRI SVVQALVLTSTVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAE

Naïve Balb/c mice (4 mice per group) were vaccinated four times s.c.(week0, week2, week4 and week6 with 2 nmol of Z13Mad8Anaxa.

To investigate the CD4 T cell responses after vaccination, one weekafter the 4^(th) vaccination, mice were euthanized; organs recovered andex vivo Elispot assay was performed on spleen cells stimulated withgp70CD4 peptide (SEQ ID NO: 64) or gp70CD8 peptide (SEQ ID NO: 65) inorder to quantify IFN-γ-producing epitope-specific CD4 and CD8 T cells.

The results are shown in FIG. 49. Mice vaccinated with Z13Mad8Anaxashowed a significant increase in IFN-γ-producing cells compared to naïvemice. These data show that Z13Mad8Anaxa vaccine was able to elicitpotent epitope-specific CD8 and CD4 T cell immune response and thus thatthe complex according to the present invention is able to elicitself-antigen immune response.

Example 32 Effect of Another Antigenic Cargo in the Complex According tothe Present Invention

To investigate the effect of a further different antigenic cargo(“Mad11”), another complex comprising a cell penetrating peptide,different antigens and a TLR peptide agonist was designed(“Z13Mad11Anaxa”). Z13Mad11Anaxa differs from Z13Mad5Anaxa (described inExample 7) in the antigenic cargoes. In particular, “Z13Mach11Anaxa” isa fusion protein comprising the cell-penetrating peptide “Z13”, theantigenic cargo “MAD11” comprising two CD8 epitopes of surviving asdescribed in Derouazi M, Wang Y, Marlu R, et al. Optimal epitopecomposition after antigen screening using a live bacterial deliveryvector: Application to TRP-2. Bioengineered Bugs. 2010;1(0:51-60.doi40.4161/bbug.1.1.9482, and the TLR peptide agonist “Anaxa”. In thefollowing, the amino acid sequence of Z13Mad11Anaxa is shown:

(SEQ ID NO: 40) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNYRIATFKNWPFLEDCAMEELTVSEFLKLDRQRSTVHEILCKLSLEGDHSTPPS AYGSVKPYTNFDAE

Naïve C57BL/6 mice (5 mice per group) were implanted i.v. with 1×10⁵ B16melanoma tumor cells and vaccinated twice (d0 and d10) by subcutaneousinjection of 1 nmol of Z13Mad11Anaxa.

On day18 mice were euthanized, organs recovered and ex vivo Elispotassay was performed on spleen cells stimulated with survivin peptidessurvivin20-28 (SEQ ID NO: 67) and survivin97-104: (SEQ ID NO: 68) inorder to quantify IFN-γ producing survivin-specific T cells.

The results are shown in FIG. 50. Mice vaccinated with Z13Mad11Anaxashowed less metastasis compared to naïve mice (FIG. 50A). Moreover, inthe spleen of mice vaccinated with Z13Mad11Anaxa significantly highernumbers of IFN-γ producing survivin-specific T cells were observed (FIG.49B).

The results obtained show that Z13Mad11Anaxa is efficacious for reducingthe number of metastasis and Z13Mad11Anaxa is able to promote thesecretion of cytokines by antigen-specific CD8 T cells in the spleen.

Example 33 Effect of Another Antigenic Cargo in the Complex According tothe Present Invention

To investigate the effect of a further different antigenic cargo(“Mad9”), another complex comprising a cell penetrating peptide, adifferent antigen and a TLR peptide agonist was designed(“Z13Mad9Anaxa”). Z13Mad9Anaxa differs from Z13Mad5Anaxa (described inExample 7) in the antigenic cargo. In particular, “Z13Mad9Anaxa” is afusion protein comprising the cell-penetrating peptide “Z13”, theantigenic cargo “Mad9” comprising the neoantigen as identified by Yadavet al. Nature. 2014 Nov 27;515(7528):572-6 from MC-38 tumor cell line,and the TLR peptide agonist “Anaxa”. In the following, the amino acidsequence of Z13Mad9Anaxa is shown with the cell-penetrating peptide“Z13” shown underlined and the TLR peptide agonist “Anaxa” shown initalics:

(SEQ ID NO: 41) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKHLELASMTNMELMSSIVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Naïve C57BL/6 mice (4 mice per group) were vaccinated four times s.c.(week0, week2, week4 and week6 with 2 nmol of Z13Mad9Anaxa. Toinvestigate the CD8 T cell responses after vaccination, one week afterthe 4th vaccination, mice were euthanized, organs recovered and Elispotassay was performed on spleen cells after a 7-day in vitro restimulationwith stimulated with adpgk peptide (SEQ ID NO: 66) in order to quantifyto quantify IFN-y-producing epitope-specific CD8 T cells.

The results are shown in FIG. 51. Mice vaccinated with Z13Mad9Anaxashowed a significant increase in effector neoantigen-specific CD8 Tcells compared to naïve mice.

Example 34 Comparison of the Immune Responses After Vaccination withComplexes Having Different Cell Penetrating Peptides

In this experiment the effect of a further different CPP in the complexaccording to the present invention was investigated by using a complexwith the TLR agonist “Anaxa”. Therefore, the fusion protein Z13Mad5Anaxaas described above (cf. Example 7, SEQ ID NO: 28) was used.

In addition, a further fusion protein was designed, which comprise theTAT CPP combined to furin linkers as described in Lu et al.,Multiepitope trojan antigen peptide vaccines for the induction ofantitumor CTL and Th immune responses J. Immunol., 172 (2004), pp.4575-4582. That fusion protein also comprises the protein “MAD5”, whichconsists of different CD8+ and CD4+ epitopes from various antigens, andthe TLR4 peptide agonist “Anaxa”. Accordingly, the following constructwas additionally designed:

TatFMad5Anaxa Sequence: (SEQ ID NO: 46)RKKRRQRRRRVKRISQAVHAAHAEINEAGRRVKRKVPRNQDWLRVKRASFEAQGALANIAVDKARVKRSIINFEKLRVKRSTVHEILCKLSLEGDH STPPSAYGSVKPYTNFDAE

C57BL/6 mice were assigned to three different groups (8 mice per group):one group receiving 2 nmol of Z13Mad5Anaxa, one group receiving 2 nmolof TatFMad5Anaxa and a respective control. The mice were vaccinated twotimes (Week0 and Week2) s.c. with either 2 nmol of Z13Mad5Anaxa or 2nmol of TatFMad5Anaxa. Mice were bled 7 days after the 2^(nd)vaccination and multimer staining was performed (8 mice per group).

The results are shown in FIG. 52. Mice vaccinated with Z13Mad5Anaxa orTatFMad5Anaxa showed an increased percentage of multimer-positive cellscompared to the control group. These results indicate that complexesaccording to the present invention having different cell penetratingpeptides are able to elicit an immune response at different doses.However, the CPP derived from ZEBRA (Z13) was better than the TAT CPP.

Example 35 Superior Efficacy of Z13Mad5Anaxa Fusion Construct Comparedto Z13Mad5 and Anaxa in Naïve Mice

Next, the efficacy of a complex according to the present invention wasinvestigated in naïve mice. Namely, Z13Mad5Anaxa (cf. Example 7; SEQ IDNO: 28) was administered to one group of mice, whereas Z13Mad5 (SEQ IDNO: 29) and Anaxa (SEQ ID NO: 15) were administered (both together) toanother group of mice.

C57BL/6 mice of the Z13Mad5Anaxa group and of the Z13Mad5+ Anaxa groupwere vaccinated once (Week0) by s.c. injection of 2 nmol of Z13Mad5Anaxa(group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa (group 2). At day 14,the blood was analyzed, whereby SIINFEKL-specific CD8 T cells werequantified in blood by multimer staining (4-8 mice per group).

Results are shown in FIG. 53. A significantly higher percentage ofSIINFEKL-specific CD8 T cells was observed in the blood ofZ13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with Z13Mad5and Anaxa separately (FIG. 53).

Taken together, these results demonstrate that Z13Mad5Anaxa vaccine (ascompared to Z13Mad5 and Anaxa administered separately) was able toelicit a stronger SIINFEKL specific CD8 T cell immune response in theperiphery.

Example 36 Effect of Another Antigenic Cargo in the Complex According tothe Present Invention

To investigate the effect of a further different antigenic cargo(“Mad12”), another complex comprising a cell penetrating peptide, adifferent antigen and a TLR peptide agonist was designed(“Z13Mad12Anaxa”). Z13Mad12Anaxa differs from Z13Mad5Anaxa (described inExample 7) in the antigenic cargo. In particular, “Z13Mad12Anaxa” is afusion protein comprising the cell-penetrating peptide “Z13”, theantigenic cargo “MAD12” comprising three neoantigens as identified byYadav et al. Nature. 2014 Nov 27;515(7528):572-6 from MC-38 tumor cellline, and the TLR peptide agonist “Anaxa”. In the following, the aminoacid sequence of Z13Mad12Anaxa is shown:

(SEQ ID NO: 69) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEGSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Naïve C57BL/6 mice (4 mice per group) were vaccinated twice s.c. (week0,week2) with 2 nmol of Z13Mad12Anaxa. To investigate the CD8 T cellresponses after vaccination, one week after the 2^(nd) vaccination, theblood was analyzed, whereby neoantigen reps1-specific CD8 T cells werequantified in blood by multimer staining (4 mice per group).

The results are shown in FIG. 54. Mice vaccinated with Z13Mad12Anaxashowed a significant increase in effector neoantigen-specific CD8 Tcells compared to naïve mice.

Example 37 In Vitro Human Dendritic Cell Maturation

The goal of this study was to investigate the capacity of a complex foruse according to the present invention (“Z13Mad5Anaxa”, SEQ ID NO: 28,cf. Example 7) to induce maturation of dendritic cells in comparison toa complex lacking a TLR peptide agonist (“Z13Mad5”, SEQ ID NO: 29, cf.Example 1).

The Z13Mad5Anaxa polypeptide and the Z13Mad5 polypeptide wereinvestigated for their capacity to induce human dendritic cell (DC)maturation. After incubation over night with 300 nM of protein,activation markers expression (CD86, CD80, CD83 and HLA-DR) was assessedon the human DCs by FACS (FIG. 55). Same buffer volumes of each proteinwere used as negative controls.

Results are shown in FIG. 55. Whereas Z13Mad5Anaxa induced maturation ofhuman DCs, shown by the up-regulation of CD86, HLADR and CD83, Z13Mad5was not able to activate human DCs. These results indicate that theAnaxa portion of the protein is responsible for the up-regulation of theactivation markers on the human DCs.

TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NOSequence Remarks SEQ ID NO: 1 RQIKIYFQNRRMKWKK CPP: PenetratinSEQ ID NO: 2 YGRKKRRQRRR CPP: TAT minimal SEQ ID NO: 3MMDPNSTSEDVKFTPDPYQVPFVQAFDQAT ZEBRA amino acidRVYQDLGGPSQAPLPCVLWPVLPEPLPQGQL sequence (naturalTAYHVSTAPTGSWFSAPQPAPENAYQAYAA sequence fromPQLFPVSDITQNQQTNQAGGEAPQPGDNST Epstein-Barr virusVQTAAAVVFACPGANQGQQLADIGVPQPAP (EBV)) (YP_401673)VAAPARRTRKPQQPESLEECDSELEIKRYKN RVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF SEQ ID NO: 4KRYKNRVASRKCRAKFKQLLQHYREVAAAK CPP1 (Z11) SSENDRLRLLLKQMC SEQ ID NO: 5KRYKNRVASRKCRAKFKQLLQHYREVAAAK CPP2 (Z12) SSENDRLRLLLK SEQ ID NO: 6KRYKNRVASRKSRAKFKQLLQHYREVAAAKS CPP3 (Z13) SENDRLRLLLK SEQ ID NO: 7KRYKNRVASRKSRAKFKQLLQHYREVAAAK CPP4 (Z14) SEQ ID NO: 8 KRYKNRVASRKSRAKFKCPP5 (Z15) SEQ ID NO: 9 QHYREVAAAKSSEND CPP6 (Z16) SEQ ID NO: 10QLLQHYREVAAAK CPP7 (Z17) SEQ ID NO: 11 REVAAAKSSENDRLRLLLK CPP8 (Z18)SEQ ID NO: 12 KRYKNRVA CPP9 (Z19) SEQ ID NO: 13 VASRKSRAKFK CPP10 (Z20)SEQ ID NO: 14 ESLKISQAVHAAHAEINEAGREVVGVG MAD5 cargoALKVPRNQDWLGVPRFAKFASFEAQG ALANIAVDKANLDVEQLESIINFEKLT SEQ ID NO: 15STVHEILCKLSLEGDHSTPPSAYGSVKPYTNF TLR2 peptide agonist DAE AnaxaSEQ ID NO: 16 DDDK enterokinase target site SEQ ID NO: 17 IEDGRfactor Xa target site SEQ ID NO: 18 LVPRGS thrombin target siteSEQ ID NO: 19 ENLYFQG protease TEV target site SEQ ID NO: 20 LEVLFQGPPreScission protease target site SEQ ID NO: 21 RX(R/K)Rfurin target site SEQ ID NO: 22 GGGGG peptidic linker SEQ ID NO: 23 GGGGpeptidic linker SEQ ID NO: 24 EQLE peptidic linker SEQ ID NO: 25 TEWTpeptidic linker SEQ ID NO: 26 MHHHHHHNIDRPKGLAFTDVDVDSIK EDAZ13Mad5IAWESPQGQVSRYRVTYSSPEDGIREL FPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTKRYKNRVASR KSRAKFKQLLQHYREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAGREV VGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFE KLTEWTGS SEQ ID NO: 27MHHHHHHSTVHEILCKLSLEGDHSTPP AnaxaZ13Mad5 SAYGSVKPYTNFDAEKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRL LLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEA QGALANIAVDKANLDVEQLESIINFEKL TEWTGSSEQ ID NO: 28 MHHHHHHKRYKNRVASRKSRAKFKQL Z13Mad5AnaxaLQHYREVAAAKSSENDRLRLLLKESLKI SQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIA VDKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLSLEGDHSTPPSAYGSVKPY

SEQ ID NO: 29 MHHHHHHKRYKNRVASRKSRAKFKQL Z13Mad5LQHYREVAAAKSSENDRLRLLLKESLKI SQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIA VDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 30MHHHHHHESLKISQAVHAAHAEINEAGR Mad5 EVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKL TEWTGS SEQ ID NO: 31MHHHHHHNIDRPKGLAFTDVDVDSIK EdaMad5 IAWESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVA LHDDMESQPLIGIQSTESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWL GVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 32 MHHHHHHESLKISQAVHAAHAEINEA Mad5AnaxaGREVVGVGALKVPRNQDWLGVPRFA KFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLSLEG DHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 33MHHHHHHKRYKNRVASRKSRAKFKQ Z14 Mad5Anaxa LLQHYREVAAAKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGV PRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVHEILC KLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 34MHHHHHHREVAAAKSSENDRLRLLLK Z18 Mad5Anaxa ESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQG ALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLSLEGDHSTPPSAY GSVKPYTNFDAE SEQ ID NO: 35 SIINFEKLSIINFEKL OVACD8 SEQ ID NO: 36 ISQAVHAAHAEINEAGR OVACD4 peptideSEQ ID NO: 37 MHHHHHHNIDRPKGLAFTDVDVDSIK EDAZ14Mad5IAWESPQGQVSRYRVTYSSPEDGIREL FPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTKRYKNRVAS RKSRAKFKQLLQHYREVAAAKESLKISQAVHAAHAEINEAGREVVGVGALKVPR NQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 38 MHHHHHHNIDRPKGLAFTDVDVDSIKEDAZ18Mad5 IAWESPQGQVSRYRVTYSSPEDGIREL FPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTREVAAAKSS ENDRLRLLLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRF AKFASFEAQGALANIAVDKANLDVEQL

SEQ ID NO: 39 KRYKNRVASRKSRAKFKQLLQHYREVA Z13Mad8AnaxaAAKSSENDRLRLLLKVTYHSPSYAYHQ FERRAILNRLVQFIKDRISVVQALVLTSTVHEILCKLSLEGDHSTPPSAYGSVKPY

SEQ ID NO: 40 KRYKNRVASRKSRAKFKQLLQHYREVA Z13Mad11AnaxaAAKSSENDRLRLLLKNYRIATFKNWPF LEDCAMEELTVSEFLKLDRQRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFD SEQ ID NO: 41KRYKNRVASRKSRAKFKQLLQHYREVAAAKS Z13Mad9AnaxaSENDRLRLLLKHLELASMTNMELMSSIVSTV HEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 42 HLELASMTNMELMSSIV Mad9 SEQ ID NO: 43 VTYHSPSYAYHQFERRAILNMad8

SEQ ID NO: 44 NYRIATFKNWPFLEDCAMEELTVSEF Mad11 SEQ ID NO: 45NIDRPKGLAFTDVDVDSIKIAWESPQGQVSR EDA YRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQST SEQ ID NO: 46RKKRRQRRRRVKRISQAVHAAHAEINEAGRR TatFMad5AnaxaVKRKVPRNQDWLRVKRASFEAQGALANIAV DKARVKRSIINFEKLRVKRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 47 MAPPQVLAFGLLLAAATATFAAAQEECVCENEpCAM YKLAVNCFVNNNRQCQCTSVGAQNTVICSKL AAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTA GVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFITSILYE NNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLI YYVDEKAPEFSMQGLKAGVIAVIVVVVIAVVAGIVVLVISRKKRMAKYEKAEIKEMGEMHREL NA SEQ ID NO: 48 GLKAGVIAV EpCAM epitopeSEQ ID NO: 49 MTPGTQSPFFLLLLLTVLTVVTGSGHASSTP MUC-1GGEKETSATQRSSVPSSTEKNAVSMTSSVLSS HSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNK PAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA PGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPG STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGST APPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVT SAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTR PAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA PGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPG STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGST APPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSI PSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLE DPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQF NQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQ CRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPA VAATSANL SEQ ID NO: 50 GSTAPPVHNMUC-1 epitope SEQ ID NO: 51 TAPPAHGVTS MUC-1 epitope SEQ ID NO: 52MGAPTLPPAWQPFLKDHRISTFKNWPFLEG survivin CACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQ FEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAKKVRRAIEQLAAMD SEQ ID NO: 53 RISTFKNWPF survivin epitopeSEQ ID NO: 54 MESPSAPPHRWCIPWQRLLLTASLLTFWNPP CEATTAKLTIESTPFNVAEGKEVLLLVHNLPQHL FGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIK SDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVS PRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSG ENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVT TITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNR TLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAA SNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPS ISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRN DARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRI NGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMI

SEQ ID NO: 55 YLSGANLNLS CEA epitope SEQ ID NO: 56 SWRINGIPQQCEA epitope SEQ ID NO: 57 MTEYKLVVVGAGGVGKSALTIQLIQNHFVDE Kirsten RasYDPTIEDSYRKQVVIDGETCLLDILDTAGQEE YSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTV DTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM SEQ ID NO: 58 VVVGAGGVGKirsten Ras epitope SEQ ID NO: 59 MPLEQRSQHCKPEEGLEARGEALGLVGAQA MAGE-A3PATEEQEAASSSSTLVEVTLGEVPAAESPDPP QSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYR AREPVTKAEMLGSVVGNWQYFFPVIFSKAFSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDG LLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQ ENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEE SEQ ID NO: 60 KVAELVHFL MAGE-A3 epitopeSEQ ID NO: 61 MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKV IL13Ralpha2NPPQDFEIVDPGYLGYLYLQWQPPLSLDHFK ECTVEYELKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEVQSSWAE TTYWISPQGIPETKVQDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDHALQCV DYIKADGQNIGCRFPYLEASDYKDFYICVNGSSENKPIRSSYFTFQLQNIVKPLPPVYLTFTRES SCEIKLKWSIPLGPIPARCFDYEIEIREDDTTLVTATVENETYTLKTTNETRQLCFVVRSKVNI YCSDDGIWSEWSDKQCWEGEDLSKKTLLRFWLPFGFILILVIFVTGLLLRKPNTYPKMIPEF FCDT SEQ ID NO: 62 LPFGFILIL13Ralpha2 epitope SEQ ID NO: 63 LFRAAQLANDVVLQIMEHLELASMTNMELM Mad12SSIVVISASIIVFNLLELEG SEQ ID NO: 64 LVQFIKDRISVVQA gp70CD4 peptideSEQ ID NO: 65 SPSYVYHQF gp70CD8 peptide SEQ ID NO: 66 ASMTNMELMadpgk peptide SEQ ID NO: 67 ATKNWPFL survivin20-28 SEQ ID NO: 68TVSEFLKL survivin97-104 SEQ ID NO: 69 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSZ13Mad12Anaxa SENDRLRLLLKLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEGSTV HEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 70 MELAALCRWGLLLALLPPGAASTQVCTGTD Her2/neuMKLRLPASPETHLDMLRHLYQGCQVVQGNL ELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPL NNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDT NRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGP KHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVG SCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIF GSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRIL HNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTA NRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNAR HCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEE GACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKY TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENV KIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDH VRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLL DIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREI PDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDL GPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSG GGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDP TVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKN GVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKG TPTAENPEYLGLDVPV

indicates data missing or illegible when filed

1. A method for treating colorectal cancer or initiating, enhancing orprolonging an anti-tumor-response in a subject in need thereofcomprising administering to the subject a complex comprising: a) a cellpenetrating peptide; b) at least one antigen or antigenic epitope; andc) at least one toll-like receptor (TLR) peptide agonist, wherein the atleast one TLR peptide agonist is a TLR2 and/or TLR4 peptide agonist; andwherein the components a)-c) are covalently linked.
 2. The methodaccording to claim 1, wherein the complex is a recombinant polypeptideor a recombinant protein.
 3. The method according to claim 1, whereinthe cell penetrating peptide (i) has a length of the amino acid sequenceof said peptide of 5 to 50 amino acids in total; and/or (ii) has anamino acid sequence comprising a fragment of the minimal domain ofZEBRA, said minimal domain extending from residue 170 to residue 220 ofthe ZEBRA amino acid sequence according to SEQ ID NO: 3, wherein,optionally, 1, 2, 3, 4, or 5 amino acids have been substituted, deleted,and/or added without abrogating said peptide's cell penetrating ability.4. The method according to claim 1, wherein the cell penetrating peptidehas an amino acid sequence comprising or consisting of an amino acidsequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14),SEQ ID NO: 8 (CPPS/Z15), or SEQ ID NO: 11 (CPPB/Z18), or a sequencevariant thereof sharing at least 90% sequence identity to SEQ ID NOs 6,7, 8 or 11 without abrogating said peptide's cell penetrating ability.5. The method according to claim 1, wherein the at least one antigen orantigenic epitope comprises or consists of at least one tumor epitope.6. The method according to claim 1, wherein the complex comprises morethan one antigen or antigenic epitope.
 7. The method according to claim5, wherein the at least one tumor epitope is an epitope of an antigenselected from the group consisting of EpCAM, HER-2, MUC-1, TOMM34, RNF43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5,COA-1, MAGE, SART and IL13Ralpha2.
 8. The method according to claim 7,wherein the complex comprises a) one or more epitopes of EpCAM orfunctional sequence variants thereof; b) one or more epitopes of MUC-1or functional sequence variants thereof; c) one or more epitopes ofsurvivin or functional sequence variants thereof; d) one or moreepitopes of CEA or functional sequence variants thereof; e) one or moreepitopes of KRas or functional sequence variants thereof; and/or f) oneor more epitopes of MAGE-A3 or functional sequence variants thereof. 9.The method according to claim 8, wherein the complex comprises a) afragment of EpCAM comprising one or more epitopes or a functionalsequence variant thereof; b) a fragment of MUC-1 comprising one or moreepitopes or a functional sequence variant thereof; c) a fragment ofsurvivin comprising one or more epitopes or a functional sequencevariant thereof; d) a fragment of CEA comprising one or more epitopes ora functional sequence variant thereof; e) a fragment of KRas comprisingone or more epitopes or a functional sequence variant thereof; and/or f)a fragment of MAGE-A3 comprising one or more epitopes or a functionalsequence variant thereof.
 10. The method according to claim 5, whereinthe at least one tumor epitope is an epitope of a neoantigen.
 11. Themethod according to claim 1, wherein the at least one TLR peptideagonist comprises or consists of an amino acid sequence according to SEQID NO: 15 or a sequence variant thereof sharing at least 90% sequenceidentity to SEQ ID NO:15 without abrogating said peptide's TLR agonistability.
 12. The method according to claim 2, wherein the components a)to c) are positioned in N-terminal→C-terminal direction of the mainchain of said complex in the order: (a) component a)—componentb)—component c); or (β) component c)—component a)—component b), whereinthe components may be linked by a further component.
 13. A method fortreating colorectal cancer or initiating, enhancing or prolonging ananti-tumor-response in a subject in need thereof comprisingadministering to the subject a nucleic acid encoding the complex asdefined in claim 1, wherein the complex is a polypeptide or a protein.14. A method for treating colorectal cancer or initiating, enhancing orprolonging an anti-tumor-response in a subject in need thereofcomprising administering to the subject a vector comprising the nucleicacid as defined in claim
 13. 15. A method for treating colorectal canceror initiating, enhancing or prolonging an anti-tumor-response in asubject in need thereof comprising administering to the subject a cellloaded with the complex as defined in claim
 1. 16. The method accordingto claim 15, wherein said cell is an antigen presenting cell.
 17. Amethod for treating colorectal cancer or initiating, enhancing orprolonging an anti-tumor-response in a subject in need thereofcomprising administering to the subject a vaccine comprising at leastone of: (i) a complex comprising a) a cell penetrating peptide; b) atleast one antigen or antigenic epitope; and c) at least one TLR2 peptideagonist and/or TLR4 peptide agonist; wherein the components a)-c) arecovalently linked; (ii) a nucleic acid encoding the complex as defined(i); (iii) a vector comprising the nucleic acid as defined in (ii); (iv)a host cell comprising the vector as defined in (iii); or (v) a cellloaded with a complex as defined in (i).
 18. A method for treatingcolorectal cancer or initiating, enhancing or prolonging ananti-tumor-response in a subject in need thereof comprisingadministering to the subject a pharmaceutical composition comprising atleast one complex comprising a) a cell penetrating peptide; b) at leastone antigen or antigenic epitope; and c) at least one TLR2 peptideagonist and/or TLR4 peptide agonist; wherein the components a)-c) arecovalently linked; and a pharmaceutically acceptable carrier.
 19. Amethod for treating colorectal cancer or initiating, enhancing orprolonging an anti-tumor-response in a subject in need thereofcomprising administering to the subject a combination of (i) a complexcomprising a) a cell penetrating peptide; b) at least one antigen orantigenic epitope; and c) at least one TLR2 peptide agonist and/or TLR4peptide agonist; wherein the components a)-c) are covalently linked; and(ii) a chemotherapeutic agent, a targeted drug and/or animmunotherapeutic agent.
 20. The method according to claim 3, whereinthe length of the amino acid sequence of said peptide is 10 to 45 aminoacids in total.
 21. The method according to claim 20, wherein the lengthof the amino acid sequence of said peptide is 15 to 45 amino acids intotal.
 22. The method according to claim 9, wherein the at least oneantigen or antigenic epitope comprises or consists of at least onecolorectal cancer epitope.
 23. The method according to claim 22, whereinthe complex comprises 3, 4, 5, 6, 7, 8, 9, 10 or more antigen orantigenic epitopes.
 24. The method according to claim 10, wherein the atleast one tumor epitope is a colorectal cancer specific neoantigen. 25.The method according to claim 12, wherein the components are linked by alinker or a spacer.
 26. The method according to claim 16, wherein saidcell is a dendritic cell.
 27. The method according to claim 19, whereinthe chemotherapeutic agent, the targeted drug and/or theimmunotherapeutic agent is an immune checkpoint modulator.